TW201142038A - Method for identifying Mycobacterium - Google Patents
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201142038 六、發明說明: 【發明所屬之技術領域】 本發明係關於一種用於鑑定分枝桿菌 spp.)之方法;特定言之,本發明係關於一種使用專一性探 針鑑疋分枝桿菌之方法。 【先前技術】 由結核桿菌群引起之結核病(tubercu】〇sis)全球每年約有 920萬新案例,並造成約n〇萬人死亡,台灣每年亦有 14,500至16,500個肺結核病例,故是全世界公共健康的大 問題。 分枝桿菌屬包括絕對致病菌 '兼性致病菌及腐生菌。目 4已分離出將近100種分枝桿菌,其中跟人類疾病相關的 有20多種’臨床上常見的如結核桿菌群 仏心⑽/〇A complex ’ MTBC)、癍瘋分枝桿菌(M」叩隱) 、潰瘍分枝桿菌(M w/ceraw)、潰瘍腫分枝桿菌(M Mwu·5"·5)及鳥分枝桿菌群(从aWwm complex)等(Murray等 人,2005,Medical Microbiology,fifth ed , ν〇1· Elsevier201142038 VI. Description of the Invention: [Technical Field] The present invention relates to a method for identifying mycobacteria spp.); in particular, the present invention relates to the use of a specific probe for the detection of mycobacteria method. [Prior Art] Tuberculosis caused by Mycobacterium tuberculosis (tubercu 〇sis) has about 9.2 million new cases every year and causes about 10,000 deaths. Taiwan has 14,500 to 16,500 cases of tuberculosis every year, so it is the world. The big problem of public health. Mycobacteria include absolute pathogenic bacteria 'facultative pathogens and saprophytic bacteria. Mesh 4 has isolated nearly 100 mycobacteria, of which more than 20 kinds of 'clinically common such as Mycobacterium tuberculosis group (10) / 〇A complex 'MTBC), Mycobacterium sinensis (M) ), Mw/ceraw, Mycobacterium tuberculosis (M Mwu·5" 5), and Mycobacterium avium (from aWwm complex), etc. (Murray et al., 2005, Medical Microbiology, fifth Ed , ν〇1· Elsevier
Mosby.)。 結核桿菌群包含以下菌種:非洲結核桿菌 (M少cohcien.wm a/Wca«謂)、牛結核桿菌 bcG (Μ· BCG)、牛結核桿菌、卡柏結核桿菌幻 、坎結核桿菌(Μ· 、邁克結核桿菌(M wz.cr<?")以Mosby.). The Mycobacterium tuberculosis group includes the following strains: Mycobacterium tuberculosis (M less cohcien.wm a/Wca«), Mycobacterium tuberculosis bcG (Μ·BCG), Mycobacterium tuberculosis, Kabo tuberculosis, Mycobacterium tuberculosis (Μ· , M. tuberculosis (M wz.cr <?")
及結核桿菌(Μ· iMZjercw/c^^KDjelouadji等人,2008,PLoSAnd Mycobacterium tuberculosis (Μ·iMZjercw/c^^KDjelouadji et al., 2008, PLoS
Neg】. Trop· Dis. 2:e253 ; Murray等人,2007,Manual of 146866.doc 201142038 CLINICAL MICROBIOLOGY,f ed,ν〇1 υ,這些菌種 彼此間的形態相近且皆會引發人類或動物結核病,其中以 非洲結核桿®、牛結核桿菌及結核桿菌較冑造成人類肺結 核病(Singleton 及 Sainsbury ’ 2〇〇6 , Dicti〇nary 〇fNeg]. Trop· Dis. 2:e253; Murray et al., 2007, Manual of 146866.doc 201142038 CLINICAL MICROBIOLOGY, f ed, ν〇1 υ, these strains are similar in shape to each other and cause human or animal tuberculosis Among them, tuberculosis rods, tubercle bacilli and tubercle bacilli in Africa cause human tuberculosis (Singleton and Sainsbury ' 2〇〇6 , Dicti〇nary 〇f
Microbiology and Molecular Bi〇I0gy,3州 ed , v〇1 ),尤以 結核桿菌為最。 正確鑑定MTBC中的菌種有助於結核病的流行病學調查 ,但因MTBC成員在基因上的相似性非常高(Sreevatsan, 人,1997,Proc· Natl. Acad. Sci. USA 94:9869-9874),在 16S rRNA基因中有99-100%的序列相似性,故此段基因的 序列並不容易區分這些菌種;近年來,用來區分MTbc的 目標基因包括尸沉」、0砂及、⑽p祁、/、幻等(Liebana 等人 ’ 1996,J, Clin. Microbiol. 34:933-938 ; Prabhakar等 人 ’ 2004 ’ J. Clin. Microbiol. 42:2724-2732 ; Scorpio 及 Zhang ’ 1996,Nat. Med. 2:662-667 ; Sreevatsan等人, 1996,J. Clin. Microbiol· 34:2007-2010),其中幻基因已 被證實是一個區分MTBC菌種的較佳基因(Chimara等人, 2004 ’ Mem. Inst. Oswald。· Cruz. 99:745-748 ; Goh等人, 2006 ’ Mol. Cell· Probes, 20:182-190 ; Niemann 等人, 2000,J. Clin. Microbiol. 38:3231-3234) o 結核桿菌群之外的分枝桿菌,一般統稱為非結核桿菌群 分枝桿菌(nontuberculous mycobacteria,NTM)。大多數的 NTM存於自然界的土壌或水中,少數則會造成人類或動物 的祠機性感染(Field 及 Cowie,2006 ’ Chest· 129:1653- I46866.doc 201142038 1 672),在免疫低下時較易感染致病,近年來臨床上NTM 的分離率有逐漸提高的趨勢(Griffith,2007,Curr. Opin. Infect Dis. 20:198-203)。由於NTM在顯微鏡底下無法與結 核桿菌區分’通常在培養分離後才能做菌種鑑定,故需要 花費較長的鑑定時間。現今有一些結合分子技術的方法, 例如:利用IS67川限制性片段長度多樣性(IS<5770-restriction fragment length polymorphism , RFLP) (Kallenius等人,1999,J· Clin. Microbiol. 37:3872-3878) 熱休克蛋白65 λκ合S#連鎖反應及限制酶分析(/^户65 PCR-restriction assay) (Telenti 等人,]993,】.(:1111· Microbiol· 31:175-178)、多位點可變數量串聯重複序列分 析(mycobacterial interspersed repetitive-unit-variable-number tandem-repeat,MIRU-VNTR)或者間隔區寡核苷酸 分型技術(spoligotyping)作分型(Aranaz 等人,2004,j·Microbiology and Molecular Bi〇I0gy, 3 states ed , v〇1 ), especially Mycobacterium tuberculosis. Correct identification of strains in MTBC contributes to the epidemiological investigation of tuberculosis, but the genetic similarity of MTBC members is very high (Sreevatsan, Ren, 1997, Proc. Natl. Acad. Sci. USA 94:9869-9874 ), there is 99-100% sequence similarity in the 16S rRNA gene, so the sequence of this gene is not easy to distinguish these species; in recent years, the target genes used to distinguish MTbc include cadaveric sinks, 0 sand and (10) p祁, /, 幻, etc. (Liebana et al. '1996, J, Clin. Microbiol. 34:933-938; Prabhakar et al.' 2004 'J. Clin. Microbiol. 42:2724-2732; Scorpio and Zhang '1996, Nat Med. 2: 662-667; Sreevatsan et al., 1996, J. Clin. Microbiol 34: 2007-2010), in which the illusion gene has been shown to be a better gene for distinguishing MTBC species (Chimara et al., 2004). ' Mem. Inst. Oswald. · Cruz. 99: 745-748; Goh et al., 2006 'Mol. Cell· Probes, 20: 182-190; Niemann et al., 2000, J. Clin. Microbiol. 38:3231- 3234) o Mycobacteria other than Mycobacterium tuberculosis, generally referred to as nontuberculous mycobacteria (nontuberculous mycoba) Cteria, NTM). Most NTMs are found in natural soil or water, and a small number can cause human or animal rickets (Field and Cowie, 2006 'Chest 129:1653- I46866.doc 201142038 1 672), when immune is low It is susceptible to infection and has a tendency to gradually increase the clinical rate of NTM in recent years (Griffith, 2007, Curr. Opin. Infect Dis. 20: 198-203). Since NTM cannot be distinguished from Rhizoctonia under the microscope, it is usually only after culture and isolation that strain identification is required, so it takes a long time to identify. There are several methods for combining molecular techniques, such as the use of IS67-restriction fragment length polymorphism (RFLP) (Kallenius et al., 1999, J. Clin. Microbiol. 37:3872-3878). Heat shock protein 65 λκ combined S# chain reaction and restriction enzyme assay (Telenti et al, 993, 】. (:1111·Microbiol· 31:175-178), multiple Point typing by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) or spacer oligonucleotide typing (Aranaz et al., 2004, j) ·
Clin. Microbiol· 42:5388-5391 ; Cadmus等人,2006,J. Clin. Microbiol. 44:29-34 ; Godreuil等人,2007,J. Clin.Clin. Microbiol·42:5388-5391; Cadmus et al., 2006, J. Clin. Microbiol. 44:29-34; Godreuil et al., 2007, J. Clin.
Microbiol. 45:921-927 ; Kremer 等人,2〇〇5,j CHn Microbiol. 43:314-320 ; Kulkarni 等人’ 2〇〇5 ,Res Micr〇bi〇L 156:588-596 ; S〇la等人,2〇〇3,以處Microbiol. 45: 921-927; Kremer et al., 2〇〇5, j CHn Microbiol. 43:314-320; Kulkarni et al. '2〇〇5, Res Micr〇bi〇L 156:588-596; S〇 La et al, 2〇〇3, everywhere
Evol. 3Π25-13 3),但臨床上有時並未對NTM做進一步的鑑 定。Evol. 3Π25-13 3), but NTM is sometimes not further clinically identified.
由於分枝桿菌生長緩慢,一般而言,臨床上一般需要2 週至8週的時間才能鑑定分枝桿菌,耗時相當長。近年來 ,發展許多的分子生物檢測法及套組,例如iNN〇 LipA 146866.doc 201142038Due to the slow growth of mycobacteria, it is generally clinically necessary to take 2 weeks to 8 weeks to identify mycobacteria, which takes a long time. In recent years, many molecular bioassays and kits have been developed, such as iNN〇 LipA 146866.doc 201142038
MYCOBACTERIA (Innogenetics,Ghent,Belgium),使用 分枝桿菌的rRNA operon作為目標序列(Suffys等人,2001 ’ J. Clin. Microbiol· 39:4477-4482 ; Tortoli等人,2003, J. Clin. Microbiol. 41:4418-4420 ; Trombert-Paolantoni 等 人,2004,Pathol. Biol. (Paris) 52:462-468),可區分出結 核桿菌群與16種非結核分枝桿菌;GenoType® Mycobacterium CM/AS (Hain Lifesience GmbH > Nehren > Germany)則使利用23 S rRNA基因作為其鑑定菌種之目標 (Makinen等人,2006,Clin. Microbiol. Infect. 12:481-483 ;Richter等人,2006,J. Clin. Microbiol. 44:1769-1775), 可區分出結核桿菌群與14種(GenoType® CM)或 16種(GenoType® Mycokcierz’Mm AS)非結核分枝桿 函,Gen-Probe Amplified Mycobacterium tuberculosis direct test (Gen-Probe,Inc.,San Diego,CA,USA)則以 1 6S rRNA為標的檢測結核桿菌群 (Chedore及Jamieson, 2002 ’ Int. J. Tuberc. Lung Dis. 6:913-919 ; Lemaitre 等人 ’ 2004,J. Clin. Microbiol. 42:4307-4309 ; Sloutsky 等人 ’ 2004 ’ J. Clin. Microbiol. 42:1547-1551);而 AmplicorTM nucleic acid amplification test (Roche Diagnostic Systems ’ Inc.,Branchburg,NJ,USA)則是利用 16S rRNA為目標 檢測結核桿菌群(Bergmann 及 Woods,1996,J. Clin. Microbiol. 34:1083-1085 ; Michos 等人'2006'Diagn. Microbiol. Infect Dis· 54:121-126)。LCx MTB assay, ABBOTT LCx probe system (Abbott Laboratories,Abbott 146866.doc • 6 - 201142038MYCOBACTERIA (Innogenetics, Ghent, Belgium), using rRNA operon of mycobacteria as the target sequence (Suffys et al, 2001 'J. Clin. Microbiol 39: 4477-4482; Tortoli et al, 2003, J. Clin. Microbiol. 41:4418-4420; Trombert-Paolantoni et al., 2004, Pathol. Biol. (Paris) 52: 462-468), distinguishes between Mycobacterium tuberculosis and 16 non-tuberculous mycobacteria; GenoType® Mycobacterium CM/AS ( Hain Lifesience GmbH > Nehren > Germany) used the 23 S rRNA gene as a target for its identification (Makinen et al., 2006, Clin. Microbiol. Infect. 12:481-483; Richter et al., 2006, J Clin. Microbiol. 44:1769-1775), distinguishes between Mycobacterium tuberculosis and 14 (GenoType® CM) or 16 (GenoType® Mycokcierz'Mm AS) non-tuberculous branching rods, Gen-Probe Amplified Mycobacterium tuberculosis Direct test (Gen-Probe, Inc., San Diego, CA, USA) detects the Mycobacterium tuberculosis group using 16S rRNA as the target (Chedore and Jamieson, 2002 ' Int. J. Tuberc. Lung Dis. 6: 913-919; Lemaitre et al. '2004, J. Clin. Microbiol. 42:4 307-4309; Sloutsky et al. '2004 'J. Clin. Microbiol. 42:1547-1551); and AmplicorTM nucleic acid amplification test (Roche Diagnostic Systems ' Inc., Branchburg, NJ, USA) targeting 16S rRNA Detection of Mycobacterium tuberculosis (Bergmann and Woods, 1996, J. Clin. Microbiol. 34: 1083-1085; Michos et al. '2006 'Diagn. Microbiol. Infect Dis. 54: 121-126). LCx MTB assay, ABBOTT LCx probe system (Abbott Laboratories, Abbott 146866.doc • 6 - 201142038
Park,111.) (Rohner 等人,1998 ’ J. Clin· Microbiol. 36:3046-3047)利用連接酶連鎖反應偵測結核桿菌群;BD ProbeTec energy transfer (ET) system (DTB) (Becton Dickinson Biosciences Microbiology Products,Sparks, MD ’ USA) (Barrett 等人,2002,J. Med. Microbiol. 51:895-898)利用IS67/0與16SrRNA基因為目標檢測結核分 枝桿菌群。 目前亦有許多研究以液態培養基BACTEC MGIT 960陽 性反應培養並確認抗酸性染色為陽性後,再結合分子生物 方法鑑定其中的分枝桿菌。包括使用基因雙套式PCR 及限制酶分析(rpoB duplex PCR-restriction enzyme analysis)、光反應性與兩種生化反應,可區分29種NTM (Shen等人,2009,Int. J. Tuberc. Lung Dis. 13:472-479)。 也有研究利用抗酸性染色為陽性之MGIT陽性反應管,進 行以IS6/川為標的的PCR反應,成功鑑定其中MTBC。由 痰塗片抗酸性染色陽性與陰性之檢測時間可分別縮短至Park, 111.) (Rohner et al., 1998 'J. Clin·Microbiol. 36:3046-3047) Detection of Mycobacterium tuberculosis using a ligase linkage reaction; BD ProbeTec energy transfer (ET) system (DTB) (Becton Dickinson Biosciences Microbiology Products, Sparks, MD 'USA) (Barrett et al., 2002, J. Med. Microbiol. 51: 895-898) The detection of Mycobacterium tuberculosis using the IS67/0 and 16SrRNA genes as targets. At present, many studies have carried out the positive reaction of liquid medium BACTEC MGIT 960 and confirmed that acid-fast staining is positive, and then combined with molecular biological methods to identify mycobacteria. Including the use of gene double-plex PCR and restriction enzyme analysis (rpoB duplex PCR-restriction enzyme analysis), photoreactivity and two biochemical reactions, can distinguish 29 NTM (Shen et al, 2009, Int. J. Tuberc. Lung Dis 13:472-479). It has also been studied to use the MGIT-positive reaction tube positive for acid-fast staining, and the PCR reaction with IS6/Chuan as the standard was successfully carried out to identify MTBC. The detection time of positive and negative anti-acid staining by sputum smear can be shortened to
6.4 天與 14.3 天(Sun 等人 ’ 2009,J. Formos. Med. Assoc. 108:119-125)。先前提到利用PCR來擴增hpM並配合RFLP 技術鑑定分枝桿菌’該技術雖已廣為使用,但在培養過程 仍要花不少時間,若直接消化經去汙染之臨床檢體,則常 因菌量不足使得敏感度下降。Chang等人結合了毛細管電 泳以及巢式聚合酶連鎖反應(nestec}-PCR)來增加/^/7(5*5及 RFLP之敏感度(Chang等人,2008,Talanta. 77:182-188)。 台灣林口長庚醫院Wu等人的研究顯示以/2^65為標的,以 146866.doc 201142038 nested-PCR和RFLP ’檢測陽性痰塗片檢體中的mtbC及部 分NTM ’有不錯的效果(Wu等人,2008,J. Clin. Microbiol. 46:3591-3594) ° 然而上述方法多有操作複雜及耗時長之缺點,且許多方 法都無法鑑定出菌種’正確率與靈敏度亦不甚令人滿意, 業界仍須開發可正確鑑定分枝桿菌之方法。 【發明内容】 本發明之目的在於提供一種鑑定分枝桿菌之方法,該方 法已括使Myc2及Myc3之楝針或其互補股(c〇mpiementary strands)與待測樣品中之DNA進行雜合反應,如有至少一 雜合反應發生,則該待測樣品包含分枝桿菌。 本發明之另一目的在於提供一種探針,其包含選自由下 列探針及其互補股所組成之群:Myc2、Myc3、Μ丁BC2、 MTBC4、MbovGl、MbovGWl、Mbovl、MbovWl、Mcanl 、McanWl、Mcapl、McapWl、融⑹、麻丨,、 MtublR、MtubWlR、Mabsl、_、μ — 3、Mf〇ri、6.4 days and 14.3 days (Sun et al. 2009, J. Formos. Med. Assoc. 108: 119-125). Previously mentioned the use of PCR to amplify hpM and identify mycobacteria with RFLP technology. Although this technology has been widely used, it still takes a lot of time in the culture process. If the decontaminated clinical specimen is directly digested, it is often The sensitivity is reduced due to insufficient bacteria. Chang et al. combined capillary electrophoresis and nested polymerase chain reaction (nestec}-PCR) to increase /^/7 (5*5 and RFLP sensitivity (Chang et al., 2008, Talanta. 77: 182-188). Wu et al., from Lin Geng Chang Gung Hospital, Taiwan, showed that mt^C and some NTMs in sputum smear samples with 146866.doc 201142038 nested-PCR and RFLP 'have good results (Wu) Et al., 2008, J. Clin. Microbiol. 46:3591-3594) ° However, the above methods are often complicated and time consuming, and many methods cannot identify the bacteria. The correct rate and sensitivity are not so Satisfactory, the industry still has to develop a method for correctly identifying mycobacteria. SUMMARY OF THE INVENTION The object of the present invention is to provide a method for identifying mycobacteria, which has included a needle of Myc2 and Myc3 or a complementary strand thereof ( C〇mpiementary strands) is heterozygously reacted with the DNA in the sample to be tested, and if at least one heterozygous reaction occurs, the sample to be tested comprises mycobacteria. Another object of the present invention is to provide a probe comprising Choose the following probes and their mutual The group consisting of: Myc2, Myc3, Kenting BC2, MTBC4, MbovGl, MbovGWl, Mbovl, MbovWl, Mcanl, McanWl, Mcapl, McapWl, fused (6), paralysis, MtublR, MtubWlR, Mabsl, _, μ-3 , Mf〇ri,
Mgas2、Mgorl、Mint2、Mkanl、Msi/lel、Mma/ul2、 Mnonl、Mperl、Mscrl、Msme3、Mszul、Mterl 及 Mxen2。 本發明之又一目的在於提供一種用於鑑定一樣品中分枝 桿菌之套組,該套組包括前述之探針。 根據本發明之方法鑑定的正確率彳目當高,縮短鑑定時間 ’並使鑑定之流程一致化’ 1容易朝向簡單化、快速化、 寿自動化可藉由正確的帛種鑑定,於臨床上施以正確的 抗生素治療。 146866.doc 201142038 【實施方式】 本發明之目的在於提供一種鑑定分枝桿菌之方法,該方 法包括使Myc2及Myc3之探針或其互補股與待測樣品中之 DNA進行雜合反應,如有至少一雜合反應發生,則該待測 樣品包含分枝桿菌。 本發明所使用之探針資訊如表1所示。 表1Mgas2, Mgorl, Mint2, Mkanl, Msi/lel, Mma/ul2, Mnonl, Mperl, Mscrl, Msme3, Mszul, Mterl and Mxen2. It is still another object of the present invention to provide a kit for identifying a mycobacterium in a sample, the kit comprising the aforementioned probe. The correct rate identified by the method of the present invention is high, shortening the identification time and making the identification process consistent. 1 Easy to simplification, rapidization, and life automation can be clinically applied by correct identification. Treat with the right antibiotics. 146866.doc 201142038 [Embodiment] The object of the present invention is to provide a method for identifying mycobacteria, which comprises heterozygous reaction of a probe of Myc2 and Myc3 or a complementary strand thereof with a DNA in a sample to be tested, if any When at least one heterozygous reaction occurs, the sample to be tested contains mycobacteria. The probe information used in the present invention is shown in Table 1. Table 1
SEQID No. 目標微生物 名稱 序列 1 分枝桿菌 Myc2 YCTGTAGTGGGCGAGAGCCGG 2 Myc3 CACACTGTTGGGTCCTGAGRCAACA 3 結核桿菌群 MTBC2 GAAAACGCCCCAACTGGT 4 MTBC4 ATGACAACAAAGTTGGCCACC 5 牛結核桿菌群 MbovGWl GGTCAACGGTGCGGTTCTGG 6 MbovGl GGTCAACGGTACGGTTCTGG 7 牛結核桿菌 MbovWl GAAGGTCTGTAACGAACAGCTGA 8 Mbovl GAAGGTCTGTAATGAACAGCTGA 9 坎結核桿菌 McanWl CGAAAGTCGTTGTGAACAAGGC 10 Mcanl CGAAAGTTGTTGTGAACAAGGC 11 卡柏結核桿菌 McapWl CCTGGCCGCTGTGATCTC 12 Mcapl CCTGGCCGCGGTGATCTC 13 邁克結核桿菌 MmicWl GGGTACGAGTGGTCTCAGGTTT 14 Mmicl GGGTATGAGTGGTCTCAGGTTT 15 結核桿菌 MtubWIR GACTTTCGAGTCGGTGGG 16 MtublR GACTTTCGCGTCGGTGGG 17 潰瘍腫結核桿菌 Mabsl CCGTGGATGGGTAGTCG 18 鳥分枝桿菌 Mavil GTTCCCGTCTGTAATGGACG 19 龜分枝桿菌 Mche3 CCCGTGGATAGGTAGTCGG 20 偶然分枝桿菌 Mforl ATTCGATTCCCCCGCCGT 21 胃分枝桿菌 Mgas2 CAGCAAGCAAGCCAGACACACTATT 22 戈登分枝桿菌 Mgorl GCCGTGAAGGGTCATCGTCT 23 胞内分枝桿菌 Mint2 GGGTGCACAACAGCGAATG 24 堪薩斯分枝桿菌 Mkanl TGGACGAAAGCCTGGTGAC 25 蘭分枝桿菌 Msi/lel TTGGTGGAGTGTGAGCCGT 26 海洋分枝桿菌 Mma/ul2 TAGTGGATGGAAGCCGGGT r 146866.doc -9- 201142038Name Sequence SEQID No. 1 target microorganism Mycobacterium Myc2 YCTGTAGTGGGCGAGAGCCGG 2 Myc3 CACACTGTTGGGTCCTGAGRCAACA 3 tuberculosis group MTBC2 GAAAACGCCCCAACTGGT 4 MTBC4 ATGACAACAAAGTTGGCCACC 5 Mycobacterium bovis group MbovGWl GGTCAACGGTGCGGTTCTGG 6 MbovGl GGTCAACGGTACGGTTCTGG 7 Mycobacterium bovis MbovWl GAAGGTCTGTAACGAACAGCTGA 8 Mbovl GAAGGTCTGTAATGAACAGCTGA 9 sill tuberculosis McanWl CGAAAGTCGTTGTGAACAAGGC 10 Mcanl CGAAAGTTGTTGTGAACAAGGC 11 Cobber tuberculosis McapWl CCTGGCCGCTGTGATCTC 12 Mcapl CCTGGCCGCGGTGATCTC 13 Mike tuberculosis MmicWl GGGTACGAGTGGTCTCAGGTTT 14 Mmicl GGGTATGAGTGGTCTCAGGTTT 15 Mycobacterium MtubWIR GACTTTCGAGTCGGTGGG 16 MtublR GACTTTCGCGTCGGTGGG 17 swollen ulcer Mabsl CCGTGGATGGGTAGTCG 18 Mycobacterium avium Mycobacterium chelonae Mavil GTTCCCGTCTGTAATGGACG 19 Mche3 CCCGTGGATAGGTAGTCGG 20 Accidental Mycobacterium Mforl ATTCGATTCCCCCGCCGT 21 Mycobacterium tuberculosis Mgas2 CAGCAAGCAAGCCAGACACACTATT 22 Mycobacterium genus Mgorl GCCGTGAAGGGTCATCGTCT 23 Mycobacterium intracellulare Mint2 GGGTGCACAACAGCGAATG 24 Mycobacterium kansii Mkanl TGGACGAAAGCCTGGTGAC 25 Mycobacterium marinum Msi/lel TTGGTGGAGTGTGAGCCGT 26 Mycobacterium marinum Mma/ul2 TAGTGGATGGAAGCCGGGT r 146866.doc -9- 201142038
27 不產色分枝桿菌 Mnonl GTGGTTGGGATACATTTCGCC 28 培力分枝桿菌 Mperl TGGTGGGTGTCGGCGTGTT 29 瘰癘分枝桿菌 Mscrl GGGGTTCTCGCCTGTAGTGG 30 猿分枝桿菌 Msi/Iel TTGGTGGAGTGTGAGCCGT 31 Msme3 TGAGGAGCTGGATCGCTGTAGT 32 桿菌 Mszul CACGAAACGCATCCCAACTG 33 土地分枯桿菌 Mterl CCGCCAGCAGCCTTGTGC 34 潰癌 —— Mma/ul2 TAGTGGATGGAAGCCGGGT 35 _餘分枯}呈钱 Mxen2 GTGTGAGTTCGGCGTGTTGT -- 特異性探斜·人# & w名方式是取分枝桿菌屬名(genus)的一個開 頭大寫英文字讲”λλ”, 于母Μ加上種名(secies)前三個小寫英文字 -^·,$灸 _$ 力 σ上揼針的序號,若為負股(anti-sense)探針會 再加上大窝念 ·· f母R。有1 9個探針選自rRNA基因間隔區 (intergenic Sna〇27 Mycobacterium sinensis Mnonl GTGGTTGGGATACATTTCGCC 28 Mycobacterium phlei Mperl TGGTGGGTGTCGGCGTGTT 29 Mycobacterium Mscrl GGGGTTCTCGCCTGTAGTGG 30 Mycobacterium Msi/Iel TTGGTGGAGTGTGAGCCGT 31 Msme3 TGAGGAGCTGGATCGCTGTAGT 32 Mycobacterium Mszul CACGAAACGCATCCCAACTG 33 Bacillus subtilis Mterl CCGCCAGCAGCCTTGTGC 34 Cancer - Mma/ul2 TAGTGGATGGAAGCCGGGT 35 _ 分 枯 呈 呈 呈 M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M ", the first three lowercase English words in the mother's name (secies) - ^ ·, $ moxibustion _ $ force σ on the needle number, if the negative (anti-sense) probe will add large Wo Nian·· f mother R. There are 19 probes selected from the rRNA gene spacer (intergenic Sna〇)
Pacer regi〇I1,its)中;另外12個探針用來區 分MTBC内的誌 J固種’設計自各菌種之幻基因特有的單一Pacer regi〇I1,its); another 12 probes are used to distinguish the unique genes unique to the phantom genes of each species in the MTBC
位點核苷酸多细M 土 I'生(single nucleotide polymorphism,SNP) ,以兩個探針兔 τ馬一組’一個是特異性探針,命名方式如前 iiji ,另一1 4固 9 n/^ $ u 了目標菌種以外的其他MTBC菌種探針, 加大寫字母W以作區別。Single nucleotide polymorphism (SNP), with two probes, a group of rabbits, a horse, a 'specific probe, named as the former iiji, another 1 4 solid 9 n/^ $ u Other probes of MTBC species other than the target strain, increase the letter W to make a difference.
^針& 4原則為該探針所在位置序列不常發生變異及: 1.探針長声人# , X "於1 7〜27個核苷酸是最理想的範圍,探針 過短會造成與目標DNA結合不易,過長容易產生非特 異性的雜合反應; 2· G C比例介於40%至60%,以降低二級結構產生的機 率; 探針:^值^ 、如elting temperature)盡可能的設計在介於雜 ,心(hybridization)時的雜合溫度(hybridization 146866.doc 201142038 temperature) 土5°C之間。例如雜合溫度若設定在5〇°c ,則探針的L值應該盡量為45°C〜55°C ; 4. 盡量避開可能會產生髮夾環結構(hairpin l〇ops)、迴 文結構(palindrome)或是重複驗基(repeats)結構; 5. 把差異點設計在整個探針的中間位置,並在探針之3, 端加入8〜10個胸腺嘧啶(thymine),以增加探針和尼龍 膜(nylon membrane)的結合; 設計完的探針經過BLAST搜索’以確定沒有與GenBankThe principle of needle & 4 is that the sequence of the probe is not often mutated and: 1. Probe long sound person # , X " in the range of 7 7~27 nucleotides is the most ideal range, the probe is too short Will cause difficulty in binding to the target DNA, too long to easily produce non-specific hybrid reactions; 2 · GC ratio between 40% and 60% to reduce the probability of secondary structure generation; Probe: ^ value ^, such as etting Temperature) The design is as close as possible to the hybridization temperature (hybridization 146866.doc 201142038 temperature) between 5 °C. For example, if the hybrid temperature is set at 5 ° ° C, the L value of the probe should be as much as 45 ° C ~ 55 ° C; 4. Try to avoid the hairpin l〇ops structure, palindrome Structure (palindrome) or repeats (repeats) structure; 5. Design the difference point in the middle of the probe, and add 8~10 thymine at the 3rd end of the probe to increase the probe. Combination of needle and nylon membrane; designed probe after BLAST search to determine no with GenBank
上其他菌種的序列相似’避免交又反應(cr〇ss hybridization) °The sequences of other strains are similar. 'cr〇ss hybridization'
本文所使用之「探針」一詞係指一包含連續至少8個核 苷酸的分子,較佳為連續10至50個核苷酸,更佳為連續15 至40個核苷酸,最佳為連續17至27個核苷酸。另一方面, 車又佳地探針之3鸠包含8至1 〇個胸腺嘧咬。該探針可與 標的DNA於雜合條件下進行雜合反應。本發明所屬技術領 域中具通常知識者可決定雜合反應之條件,其中該雜八反 應之較佳條件係於約4 〇。〇至約6 5。〇中進行。 本文所使用之「 針雜合之核酸分子 全互補之核酸分子 互補股」一詞係指可與根據本發明之探 車乂佳係可與根據本發明之探針鹼基完 根據本發明之探針可用 ^釔疋刀枝才干函,由於分枝桿! w遍生長緩慢,用傳統的 。奮居而要車乂長的時間,且部$ 的分枝桿菌不易鍟定。券 别研九才曰出,細菌的16S rRNAi 23S rRNA二個基因間隔 W £在序列上具有種的特異七 】46866.doc 201142038 (species-specific),有時比利用16S rRNA基因定序法更能 區分至種的層次。且rRNA基因間隔區序列在種内不同菌 株間的皮異性报小(Gurtler及Stanisich, 1996 , Microbiology· 142 (Pt 1):3_16),因此,本發明主要利用 rRNA基因間隔區作為分枝桿菌鑑定之目標。 較佳地,根據本發明之方法可進一步區分結核桿菌群與 非結核分枝桿菌群,其另包含使用MTBC2& mtbc4之探 針或其互補股與該待測樣品中之DNA進行雜合反應,如該 待測樣品中之DNA同時與MTBC2及MTBC4之探針或其互 補股產生雜合反應,則該待測樣品包含結核桿菌群;如該 待則‘ 中之DNA未同時與MTBC2及MTBC4之探針或其互 補版產生雜合反應,則該待測樣品包含非結核分枝桿菌。 曰更佳地,根據本發明之方法可進一步針對已鑑定為結核 杯菌群之待鑑疋菌鑑定出其種名。由於結核桿菌群的 rRNA基因間隔區具1〇〇%的相似度故利用町队在^^ 基因中有多個單一位點核誓酸多型性做該群中不同菌種的 鑑定。 據本毛明之方法較佳另包含使用及MtubwiR 之^針或其互補股與待測樣品中之DNA進行雜合反應,如該 待測樣品中之DNA與MtublR或其互補股雜合反應之強度, 大於该待測樣品中<DN^MtubwlR或其互補股雜合反應 之強度,則該待測樣品包含結核桿菌(M⑽⑽“)。 根據本發明之方法較佳另包含使用MbovGl及MbovGWl 之探針或其互補股與該待測樣品中之DNA進行雜合反應, 146866.doc 201142038 如該待測樣品_之DNA* MbovG1或其互補股雜合反應之 強度,大於該待測樣品申之DNA與MbovGWl或其互補股 雜合反應之強度,則該待測樣品包含牛結核桿菌群(从 办⑽以group)。較佳地,該牛結核桿菌群包含牛結核桿菌 (M hvb) '牛結核桿菌BCG(M 0〇WiBCG)及卡柏結=桿 菌(M. 於本發明之一較佳具體實施例中,可鑑: 出牛結核桿菌群之個別菌種。根據本發明之方法其較2 另包含使用Mbov 1及MbovWl之探針或其互補股與該待測 樣品中之DNA進行雜合反應,如該待測樣品中之dna與 Mb〇vl或其互補股雜合反應之強度’大於該待測樣品中2 讓與或其互補股雜合反應之強度,則該待測樣 品包含牛結核桿菌或牛結核桿菌BCG ;根據本發明之方法 ’其較佳3包含使用Mcapl及McapW1之探針或其互補俨 與該待測樣品中之DNA進行雜合反應,如該待測樣品中2 囊與MCapl或其互補股雜合反應之強度,大於該待測樣 品中之DNA與McapW1或其互補股雜合反應之強度,則該 待測樣品包含卡柏結核桿菌。 根據本發明之方法,其較佳另包含使用Mmicl及As used herein, the term "probe" refers to a molecule comprising at least 8 nucleotides in succession, preferably 10 to 50 nucleotides in length, more preferably 15 to 40 nucleotides in length, optimal. It is 17 to 27 nucleotides in succession. On the other hand, the car also has 8 to 1 thymidine bite. The probe can be hybridized with the target DNA under heterozygous conditions. Those of ordinary skill in the art to which the present invention pertains can determine the conditions of the hybrid reaction, wherein the preferred conditions for the heterocycle are about 4 Torr. 〇 to about 6 5 . In the middle of the battle. The term "a nucleic acid molecule complementary strand of a fully hybrid nucleic acid molecule" as used herein refers to a base which can be used with the probe according to the present invention and the base of the probe according to the present invention. Needle can be used to make a knife, because of the branching rod! w grows slowly, using traditional ones. It takes a long time to get into the house, and the mycobacteria of the Ministry is not easy to determine. The vouchers are not only developed, but the bacterial 16S rRNAi 23S rRNA two gene intervals W £ have a specificity in the sequence of seven] 46866.doc 201142038 (species-specific), sometimes more than the 16S rRNA gene sequencing method Can be distinguished to the level of the species. Moreover, the rRNA gene spacer sequence has a small susceptibility between different strains in the species (Gurtler and Stanisich, 1996, Microbiology 142 (Pt 1): 3-16). Therefore, the present invention mainly utilizes the rRNA gene spacer as a mycobacterial identification. The goal. Preferably, according to the method of the present invention, the Mycobacterium tuberculosis group and the non-tuberculous mycobacterial group can be further distinguished, which further comprises using a probe of MTBC2 & mtbc4 or a complementary strand thereof to carry out a heterozygous reaction with the DNA in the sample to be tested, If the DNA in the sample to be tested simultaneously produces a hybrid reaction with the probes of MTBC2 and MTBC4 or their complementary strands, the sample to be tested contains Mycobacterium tuberculosis; if the DNA in the sample is not simultaneously with MTBC2 and MTBC4 The probe or its complementary version produces a hybrid reaction, and the sample to be tested contains non-tuberculous mycobacteria. More preferably, the method according to the invention further identifies the species name of the toadstool that has been identified as a tuberculosis group. Since the rRNA gene spacer of the Mycobacterium tuberculosis group has a similarity of 1%, the town team has a plurality of single sites in the ^^ gene to identify the different strains in the group. According to the method of the present invention, it is preferred to carry out a heterozygous reaction with the DNA of the sample to be tested and the complementary strand of the MtubwiR, such as the strength of the heterozygous reaction between the DNA in the sample to be tested and MtublR or its complementary strand. And greater than the intensity of the heterozygous reaction of <DN^MtubwlR or its complementary strand in the sample to be tested, the sample to be tested comprises Mycobacterium tuberculosis (M(10)(10)"). The method according to the present invention preferably further comprises the use of MbovGl and MbovGWl The needle or its complementary strand is heterozygously reacted with the DNA in the sample to be tested, 146866.doc 201142038 If the DNA of the sample to be tested _ DNA* MbovG1 or its complementary strands is greater than the DNA of the sample to be tested The strength of the heterozygous reaction with MbovGW1 or its complementary strands, the sample to be tested comprises a population of Mycobacterium tuberculosis (from the group (10) to group). Preferably, the group of Mycobacterium tuberculosis comprises Mycobacterium tuberculosis (M hvb) 'bovine tuberculosis Bacillus BCG (M 0〇WiBCG) and Kappa knot = bacillus (M. In a preferred embodiment of the present invention, it is possible to identify: an individual strain of the Mycobacterium tuberculosis group. According to the method of the present invention, it is 2 Also includes probes using Mbov 1 and MbovWl or their complements Performing a hybrid reaction with the DNA in the sample to be tested, such as the intensity of the heterozygous reaction of dna with Mb〇vl or its complementary strand in the sample to be tested is greater than 2 in the sample to be tested or its complementary strand The intensity of the reaction, the sample to be tested comprises Mycobacterium tuberculosis or Mycobacterium tuberculosis BCG; the method according to the invention preferably comprises using a probe of Mcapl and McapW1 or a complementary ruthenium thereof and DNA in the sample to be tested. a heterozygous reaction, such as the intensity of the heterozygous reaction of the 2 capsule with MCap1 or its complementary strand in the sample to be tested is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with McapW1 or its complementary strand, then the sample to be tested comprises Tuberculosis bacillus. According to the method of the present invention, it preferably further comprises using Mmicl and
MmiCW1之探針或其互補股與該待測樣品中之驗進行雜 合反應’如該待測樣品中之DNA與心⑹或其互補股雜人 反應之強度,大於該待測樣品中之dna與編_或复互 補股雜合反應之強度,則該待測樣品包含邁克結核桿菌 (M. microti)。 根據本發明之方法,复如σ “ 其車乂佳另包含使用Mcani及 146866.doc •13- 201142038 之探針或其互補股與該待測樣品中 合反應,如該待測樣品令之職 進-雜 反應之诒许丄 /、Mcan】或其互補股雜合 … 又㈣待測樣口口口令之DNA與McanWl或苴互 汉應之強度,則該待測樣品包含坎結 canettii)。 τ _The probe of MmiCW1 or its complementary strand is hybridized with the test in the sample to be tested, 'if the DNA in the sample to be tested reacts with the heart (6) or its complementary strands, the intensity is greater than the dna in the sample to be tested The sample to be tested contains M. microti, which is the intensity of the heterozygous reaction with the chimeric or complementary strand. According to the method of the present invention, the sample of the sample to be tested is combined with the probe or the complementary strand thereof using Mcani and 146866.doc • 13-201142038, such as the sample to be tested. In addition to the heterozygous reaction, 丄 、 /, Mcan 】 or its complementary strands of heterozygosity ... (4) the DNA of the mouth sample to be tested and the strength of McanWl or 苴 汉 汉 , 则 则 则 则 can can can can can can can can can can can can can can can can can can τ _
互2據本發明之方法,其較佳另包含使用第—群探針或其 ί月又及第_群探針或其互補股與該待測樣品中之⑽八進 行雜合反應’如該待測樣品中之職與第:群探針或其互 補股雜合反應之強度,皆大於該待測樣品中之DNA與第一 群探針或其互補股雜合反應之強度,則該待測樣品包含非 W結核桿g (M咖·⑽謂),其中該第—群探針包含 MtublR、MbovG1、Mb〇vl、心叫 ' MmicaMcani ;第 二群探針包含 MtubW1R、MbovGW1、Mb()vW1、Meapwi 、MmicWl 及 McanWl。 另一方面,更佳地,根據本發明之方法可進一步針對已 鑑定為非結核分枝桿菌之待鑑定菌鑑定出其種名,其主要 利用rRNA基因間隔區所設計之探針而進行。 根據本發明之方法,其較佳另包含使用一或多個選自According to the method of the present invention, preferably, the method further comprises: using the first-group probe or the λ and the _ group probe or the complementary strand thereof to carry out a heterozygous reaction with the (10) VIII in the sample to be tested. The intensity of the heterozygous reaction between the sample in the sample to be tested and the group probe or its complementary strand is greater than the intensity of the heterozygous reaction between the DNA in the sample to be tested and the first group of probes or their complementary strands. The sample contains a non-W tuberculosis rod g (M coffee · (10) said), wherein the first group probe comprises MtublR, MbovG1, Mb〇vl, the heart is called 'MmicaMcani; the second group probe contains MtubW1R, MbovGW1, Mb() vW1, Meapwi, MmicWl, and McanWl. On the other hand, more preferably, the method according to the present invention can further identify the species name of the strain to be identified which has been identified as non-tuberculous mycobacteria, which is mainly carried out using a probe designed by the rRNA gene spacer. Preferably, the method according to the present invention further comprises using one or more selected from the group consisting of
Mabsl、Mavil、Mche3、Mforl、Mgas2、Mgorl、Mint2 、Mkanl、Msi/lel、Mma/ul2、Mnonl、Mperl、Mscrl、Mabsl, Mavil, Mche3, Mforl, Mgas2, Mgorl, Mint2, Mkanl, Msi/lel, Mma/ul2, Mnonl, Mperl, Mscrl,
Msme3、Mszul、Mterl、Mxen2及其互補股之探針與該待 測樣品中之DNA進行雜合反應; 如該該待測樣品中之DNA與Mabsl探針或其互補股產生 雜合反應’則該待測樣品包含潰瘍腫分枝桿菌 146866.doc 201142038 abscessus^); 人如4待測樣品中之DNA與Mavil探針或其互補股產生雜 。反應,則該待測樣品包含鳥分枝桿菌(Μ. ; 人如該待測樣品中之DNA與Mche3探針或其互補股產生雜 口反應,則該待測樣品包含龜分枝桿菌(M ; 八如該待測樣品中之〇!^八與Mf〇rl探針或其互補股產生雜 口反應’則該待測樣品包含偶‘然分枝桿菌(M細心㈣;Msme3, Mszul, Mterl, Mxen2 and their complementary strand probes are heterozygous for the DNA in the sample to be tested; if the DNA in the sample to be tested is heterozygous with the Mabsl probe or its complementary strands' The sample to be tested comprises M. ulcerans 146866.doc 201142038 abscessus^); the DNA in the human sample such as 4 is heterozygous with the Mavil probe or its complementary strand. In response, the sample to be tested comprises Mycobacterium avium (Μ.; human if the DNA in the sample to be tested reacts with the Mche3 probe or its complementary strand, the sample to be tested comprises Mycobacterium marinum (M) ; as in the sample to be tested, 八8 and the Mf〇rl probe or its complementary strands produce a miscellaneous reaction', then the sample to be tested contains the genus Mycobacterium (M careful (4);
A如該待測樣品中之DNA與^^382探針或其互補股產生雜 合反應,則該待測樣品包含胃分枝桿菌(M A如孩待測樣品中之DNA與Mg〇rm針或其互補股產生雜 。反應,則該待測樣品包含戈登分枝桿菌(从別“⑽以); 八如忒待測樣品中之DNA與]^1加2探針或其互補股產生雜 合反應’則該待測樣品包含胞内分枝桿菌(M intracelluiare^ . 如該待測樣品中之DNA#Mkanl探針或其互補股產生雜合 反應Μ 4待測樣品包含堪薩斯分枝桿® (Λ/. (_咖7); 八如該待測樣。S中之DNA與Msi/lel探針或其互補股產生雜 〜則°亥待測樣品包含猿分枝桿菌(Λ/· hwke)或蘭分 枝桿菌(M !⑽i/7avum);If the DNA in the sample to be tested is heterozygous with the ^382 probe or its complementary strand, the sample to be tested comprises Mycobacterium tuberculosis (MA such as DNA in a sample to be tested and a Mg〇rm needle or The complementary strands produce a heterogeneous reaction. The sample to be tested contains Mycobacterium gordonii (from "10"); the DNA in the sample to be tested is mixed with 2 probes or 2 complementary probes or their complementary strands. The reaction sample contains the intracellular mycobacteria (M intracelluiare^. If the DNA#Mkanl probe or its complementary strand in the sample to be tested produces a hybrid reaction Μ 4 sample to be tested contains Kansas branching rod® (Λ/. (_咖7); Eight as the sample to be tested. The DNA in S and the Msi/lel probe or its complementary strands produce a heterogeneous ~ then the sample to be tested contains Mycobacterium sputum (Λ/· hwke ) or M. kawaii (M !(10)i/7avum);
2。玄待測樣品中之DNA與Mma/ul2探針或其互補股產生 雜合反應’則該待測樣品包含海洋分枝桿菌(M祕以― 或潰癌分枝桿菌(M2. The DNA in the sample to be tested is heterozygous for the Mma/ul2 probe or its complementary strand. The sample to be tested contains Mycobacterium marinum (M-mycosis or Mycobacterium smegmatis (M)
人如該待測樣品中之DNA與Mnonl探針或其互補股產生雜 合反應,則該待測樣品包含不產色分枝桿菌(M 146866.doc 15 201142038 n〇nchr〇m〇genicum); 如該待測樣品中之DNA與Mperl探針或其互補股產生雜合 反應,則該待測樣品包含培力分枝桿菌(M ; 如該待測樣品中之DNA與Mscr!探針或其互補股產生雜 〇反應,則該待測樣品包含瘰癘分枝桿菌(从 scrofulaceum); 如邊待測樣品中之DNA與Msme;3探針或其互補股產生雜合 反應’則邊待測樣品包含耳心垢分枝桿菌(M.鹰; # 如該待測樣品中之DNA與Mszul探針或其互補股產生雜 。反應,則该待測樣品包含斯氏分枝桿菌(从η"/”,); 如該待測樣品中之DNA與MterUf針或其互補股產生雜 合反應’則該待測樣品包含土地分枝桿菌(M);及 ▲如該待測樣。〇〇中之麗八與河則以菜針或其互補股產生雜 合反應,則該待測樣品包含料分枝桿g(M㈣零.)。 根據本發明之待測樣品可為包含待鑑定菌之培養物、取 籲病人之檢體,或是自待鑑定菌培養物或自病人檢體進一 步處理以取得其中DNA資訊之樣品,丨中病人之檢體較佳 係為痰或血液,取得DNA資訊之方法較佳係藉由聚合酶連 鎖(polymerase chain reaction)反應擴增。 由於本發明之方法係藉由rRNA基因間隔區或基因 设=之探針進行鑑定’故該待測樣品中之舰較佳包含待 L ^菌之rRNA基因間隔區或基因;更佳地,該待測 樣品中之DNA包含由聚合酶連鎖反應擴增之片段。於本發 月之較佳具體實施例中,待測樣品中之dna係使用印】 146866.doc •16- 201142038 (SEQ ID NO. 36)及sp2 (SEQ ID N〇 37)引子擴增待鑑定菌 之ANA基因間隔區;待測樣品中待鑑定菌之幻^5基因較 佳係藉由 Gblf (SEQ ID Ν〇. 38)及 GMr (SEQ ⑴ ν〇 39)引 子擴增獲得。 於本發明之—較佳具體實施例中,可對樣品中DNA實施 標記。DNA之標記方法’已為本發明所屬技術領域中具通 常知識者所熟知。例如,當實施聚合酶連鎖反應擴增時, *亥引子係包含一標幟,較佳地,該標幟係為毛地黃素 ♦ (d—in),或經標記之dUTp可用於將一標記引入產物。 在本發明一較佳實施例中,該方法進一步包括一陽性對 照步驟。任何用於鑑定一已知微生物之確定探針皆適用於 此陽性對照步驟。 較佳地’根據本發明雜合反應可於一微陣列晶片 (miCroarray)上進行,該微陣列晶片包含一基材,且該探針 係塗佈於該基材上。本領域已良好地建立了基材材料及塗 籲 佈方法,其中該基材較佳係為尼龍或玻璃。 根據本發明之探針較佳以微陣列方式佈置在該基材上, 以便一次操作可鑑定多種微生物,節省大量時間費用、 空間及人力。該技術中任何常用之基因表現檢測方法皆可 用於本發明,其可取代習知技術中針對不同分枝桿菌進行 不同之生物化學反應來鑑定菌種。於本發明之一具體實施 例中’以寡核¥酸晶片(〇lig〇nucieotide array)鐘定分枝桿 菌,包括6種屬於結核桿菌群〇〇1^丨以)的 細菌。由細菌的MS-23S rRNA基因間的内轉錄區域及奵叻 146866.doc 201142038 基因設計探針,將此探針以微矩陣點製機點佈於尼龍薄膜 (nylon membrane)上,與標識毛地黃素的pcR產物進行雜 〇反應後,再以標記磷酸分解酶的抗體呈現反應結果。 b曰片先測6式模式菌株(type strain)以及參考菌株 (reference strain),再進一步測試臨床菌株最後直接測 試臨床檢體,以評估該晶片用於鑑定(或檢測)分技桿菌之 可行I·生本發明共測試目標參考菌株25種,包括MTBC ό 種(15株)(表2),ΝΤΜ 19種(73株)(表3),共88株。另測試 目標臨床菌株,其中MTBC 8〇株(表2),ΝΤΜ 121株,共 201 株(表 3)。 根據本發明之晶片可在8小時内完成菌種鑑定,且試劑 用量較少、成本較低廉、靈敏度及特異性高。故本發明之 微陣列晶片可快速及正確地鑑定不同的分枝桿菌菌種’為 一種有效的檢測工具。 本發明之另一目的在於提供一種探針,其包含選自由下 列序列所組成之群:Myc2、Myc3、MTBC2、MTBC4、If the DNA in the sample to be tested is heterozygously reacted with the Mnon1 probe or its complementary strand, the sample to be tested comprises Mycobacterium fortunei (M 146866.doc 15 201142038 n〇nchr〇m〇genicum); If the DNA in the sample to be tested produces a hybrid reaction with the Mper1 probe or its complementary strand, the sample to be tested comprises Mycobacterium phlei (M; such as the DNA in the sample to be tested and the Mscr! probe or The complementary strands produce a heterozygous reaction, and the sample to be tested comprises Mycobacterium phlei (from scrofulaceum); if the DNA in the sample to be tested and the Msme; 3 probe or its complementary strands produce a hybrid reaction, then the test is to be tested The sample comprises Mycobacterium smegmatis (M. Eagle; # if the DNA in the sample to be tested is heterozygous with the Mszul probe or its complementary strand. The reaction, the sample to be tested comprises Mycobacterium tuberculosis (from η"/",); if the DNA in the sample to be tested produces a hybrid reaction with the MterUf needle or its complementary strands', the sample to be tested contains Mycobacterium phlei (M); and ▲ as the sample to be tested. The Li Ba and the river produce a hybrid reaction with the vegetable needle or its complementary strand, and the sample to be tested contains the material branching rod g (M(四)零.) The sample to be tested according to the present invention may be a culture containing the culture to be identified, a sample taken from the patient, or further processed from the culture to be identified or from the patient to obtain DNA information therein. For the sample, the sample of the patient in the sputum is preferably sputum or blood, and the method for obtaining DNA information is preferably amplified by a polymerase chain reaction. Since the method of the present invention is separated by the rRNA gene The region or the gene set = the probe is identified. Therefore, the ship in the sample to be tested preferably contains the rRNA gene spacer or gene of the L ^ bacteria; more preferably, the DNA in the sample to be tested contains the linkage by the polymerase Reaction-amplified fragment. In a preferred embodiment of the present month, the dna in the sample to be tested is printed using 146866.doc •16-201142038 (SEQ ID NO. 36) and sp2 (SEQ ID N〇37) The primer is used to amplify the ANA gene spacer region of the strain to be identified; the magical 5 gene to be identified in the sample to be tested is preferably extended by Gblf (SEQ ID Ν〇. 38) and GMr (SEQ (1) ν〇39). Addition. In the preferred embodiment of the invention, the sample can be DNA-implemented labeling. The method of labeling DNA' is well known to those of ordinary skill in the art to which the present invention pertains. For example, when performing polymerase chain reaction amplification, the label contains a flag, preferably, The marker is digoxigenin ♦ (d-in), or the labeled dUTp can be used to introduce a label into the product. In a preferred embodiment of the invention, the method further comprises a positive control step. Any identifying probe for identifying a known microorganism is suitable for this positive control step. Preferably, the hybridization reaction according to the present invention can be carried out on a microarray wafer comprising a substrate onto which the probe is applied. Substrate materials and coating methods have been well established in the art, wherein the substrate is preferably nylon or glass. The probes according to the present invention are preferably arranged on the substrate in a microarray manner to allow identification of a plurality of microorganisms in a single operation, saving a lot of time, space and labor. Any of the commonly used gene expression detection methods in the art can be used in the present invention, which can replace the different biochemical reactions for different mycobacteria in the prior art to identify the species. In a specific embodiment of the present invention, 'bacteria are counted in a 〇lig〇nucieotide array, including six species belonging to the Mycobacterium tuberculosis group. From the internal transcribed region between the MS-23S rRNA gene of the bacterium and the 奵叻146866.doc 201142038 gene design probe, the probe was spotted on a nylon membrane by a micro-matrix dot machine, and the marker was ground. After the heterologous reaction of the pcR product of flavin, the reaction result is shown by an antibody labeled with a phosphate decomposing enzyme. b The sputum is first tested for the type strain and the reference strain, and further tested the clinical strain and finally directly tested the clinical specimen to evaluate the feasibility of the wafer for identifying (or detecting) the bacillus · The present invention tested a total of 25 target reference strains, including MTBC (15 strains) (Table 2) and 19 strains (73 strains) (Table 3), a total of 88 strains. The target clinical strains were also tested, including MTBC 8 strain (Table 2) and 121 strains, a total of 201 strains (Table 3). The wafer according to the present invention can complete the strain identification within 8 hours, and the reagents are used in a small amount, the cost is low, and the sensitivity and specificity are high. Therefore, the microarray wafer of the present invention can quickly and correctly identify different mycobacterial species' as an effective detection tool. Another object of the present invention is to provide a probe comprising a group selected from the group consisting of Myc2, Myc3, MTBC2, MTBC4,
MbovGl、MbovGWl、Mbovl、MbovWl、Mcanl、 McanWl、Mcapl、McapWl ' Mmicl、MmicWl、MtublR 、MtubWlR、Mabsl、Mavil、Mche3、Mforl、Mgas2、 Mgorl、Mint2、Mkanl、Msi/lel、Mma/ul2、Mnonl、 Mperl、Mscrl、Msme3、Mszul ' Mterl、Mxen2及其互補 股。 本發明之又一目的在於提供一種用於鑑定一樣品中分枝 桿菌之套組,該套組包括前述之探針。 f46866.doc -18- 201142038 根據本發明,該套組進—步包括用於獲得侧a基因間 隔區或#基因之試劑。該試劑之組成亦為本發明所屬技 術領域中具通常知識者所熟知。在本發明—較佳實施例中 ’试劑係用於聚合酶連鎖反應。在本發明—更佳實施例中 ,該套組進一步包括spl與sp2引子及/或(31)1£與(^^引子。 在本發明另-較佳實施例中,該套組進—步包括雜合反應 用試劑。 效以下列實例予以詳細說明本發明,唯其並不意味本發 明僅侷限於此等實例所揭示之内容。 實例: 實驗用菌株、菌株的培養、及菌種£^八 本研究所使用的參考菌株分別從美國ATCC (AmericanMbovGl, MbovGWl, Mbovl, MbovWl, Mcanl, McanWl, Mcapl, McapWl 'Mmicl, MmicWl, MtublR, MtubWlR, Mabsl, Mavil, Mche3, Mforl, Mgas2, Mgorl, Mint2, Mkanl, Msi/lel, Mma/ul2, Mnonl, Mperl, Mscrl, Msme3, Mszul 'Mterl, Mxen2 and their complementary strands. It is still another object of the present invention to provide a kit for identifying a mycobacterium in a sample, the kit comprising the aforementioned probe. F46866.doc -18- 201142038 In accordance with the present invention, the kit further comprises reagents for obtaining the lateral a gene compartment or the # gene. The composition of the reagents is also well known to those of ordinary skill in the art to which the invention pertains. In the present invention - the preferred embodiment - the reagents are used in the polymerase chain reaction. In a preferred embodiment of the invention, the kit further comprises spl and sp2 primers and/or (31) 1 £ and (^^ primers. In another preferred embodiment of the invention, the kit is advanced The invention is described in detail by the following examples, which are not intended to limit the invention to the disclosure of the examples. Examples: Experimental strains, strain cultures, and strains £^ The reference strains used in the eight research institutes are from the United States ATCC (American
Type Culture Collection ’ Manassas,Virginia,USA)、台 >4 BCRC (Bioresources Collection and Research Center,Type Culture Collection ’ Manassas, Virginia, USA), Taiwan > 4 BCRC (Bioresources Collection and Research Center,
Food Industry Research and Development Institute »Food Industry Research and Development Institute »
Hsinchu ’ Taiwan)、瑞典 CCUG (Culture Collection, University of G0teborg,G6teborg,Sweden)、法國 CIP (Collection of the Institute Pasteur,rue du Docteur Roux, Paris ’France)、日本 JCM (Japan Collection of Microorganism > RIKEN BioResource Center 1 Saitama >Hsinchu 'Taiwan', CCUG (Culture Collection, University of G0teborg, G6teborg, Sweden), CIP (Collection of the Institute Pasteur, rue du Docteur Roux, Paris 'France), Japan JCM (Japan Collection of Microorganism > RIKEN BioResource Center 1 Saitama >
Japan)、英國 NCTC (National Collection of Type Cultures, Central Public Health Laboratory,London,UK)所構得。 本研究所使用的目標參考菌株總共有25種(88株)分枝桿 菌,包括MTBC6種(15株)(表2),NTM有19種(73株)(表3) 146866.doc 19 201142038 ;另測試目標臨床菌株(clinical strains),其中MTBC 80株 (表 2),NTM 121株,共 201 株(表 3)。非目標菌(nontarget species)中屬於分枝桿菌屬之細菌有37種(38株),其他細菌 35種(35株)(表 4)。 表2 菌種 非洲結核桿菌 牛結核ϋ~ 牛結核桿菌 BCG 坎結核彳# 卡柏結核桿菌 邁克結 結核桿菌 結核桿菌群 總菌株 參考菌株 臨床菌株 總菌 株數 ATCC 25420 » ATCC 35711 ATCC 27291 » ATCC 35734 ATCC 35731 > ATCC 35735 ATCC 35737 ATCC BAA-824 ATCC 19422 ATCC 35782 NCTC 8337 H37Rv,ATCC 25177, ATCC 35818 » ATCC 35828 MB 156,MB 217,KMUH 】8709, KMUH 19297,KMUH 18702,KMUH 19149,KMUH 18886,KMUH 16758, KMUH 17123 » KMUH 17529 ' KMUH 17641,KMUH 18352,KMUH 16704, KMUH 17006 > KMUH 15546 * KMUH 14687,KMUH 16793,KMUH 15569, KMUH 14384 * KMUH 14821 » KMUH 15627,KMUH 15874,KMUH 12528, KMUH 13025,KMUH 13115,KMUH 12961,KMUH 13638,KMUH 13790, KMUH 14288,KMUH 14303,KMUH 14494,KMUH 14685,KMUH 1432 卜 KMUH 14551 > KMUH 14637 » KMUH 14155,KMUH 1469卜 KMUH 9880, KMUH 11037 > KMUH 12087 * KMUH 1118 卜 KMUH 12566,KMUH 12569, KMUH 13295 * KMUH 12088 » KMUH 9798,KMUH 9388,KMUH 9776, KMUH 8222 » KMUH 8127 » KMUH 6754,KMUH 6828,NCKUH 7290, NCKUH 7161 » NCKUH 7910 » NCKUH 7068,NCKUH 7096,NCKUH 7885, NCKUH 7476,NCKUH 7590,AUH 2 ,AUH 3,AUH 4,AUH 5,AUH 7, AUH 丨丨 * AUH 12,AUH 15,AUH 16 > AUH 20 * AUH 21 > AUH 22 * AUH 23,AUH 24,AUH 25,AUH 26,AUH 27,AUH 28,AUH 30,AUH 31 2 15 95 •20- 146866.doc 201142038 表3_ 菌種 參考i株 臨床菌株 總菌株數目 潰癌腫分枝桿菌 CCUG 20993,CCUG 37454 AUH8,KMUHT13769,KMUHT13928, 17 ,NCTC 10269,ATCC KMUHT12727 > KMUHT14817 > KMUHT14298 23003 - KMUH0729-31 - KMUH0903-49 - KMUH0903-51 > KMUH0903-92 > KMUH0903-48,KMUH0729-33,KMUH0903-96 鳥分枝桿菌Japan), British NCTC (National Collection of Type Cultures, Central Public Health Laboratory, London, UK). The target reference strains used in this study have a total of 25 (88 strains) of mycobacteria, including 6 MTBC (15 strains) (Table 2), and 19 NMS (73 strains) (Table 3) 146866.doc 19 201142038; The target clinical strains were also tested, including MTBC 80 strain (Table 2) and NTM 121 strain, a total of 201 strains (Table 3). Among the nontarget species, there were 37 species (38 strains) belonging to the genus Mycobacterium, and 35 strains (35 strains) of other bacteria (Table 4). Table 2 Mycobacterium tuberculosis bovine tuberculosis ϋ~ Bovine tuberculosis BCG TB tuberculosis 卡 #卡柏 TB Mycobacterium tuberculosis Mycobacterium tuberculosis total strain reference strain clinical strain total strain number ATCC 25420 » ATCC 35711 ATCC 27291 » ATCC 35734 ATCC 35731 > ATCC 35735 ATCC 35737 ATCC BAA-824 ATCC 19422 ATCC 35782 NCTC 8337 H37Rv, ATCC 25177, ATCC 35818 » ATCC 35828 MB 156, MB 217, KMUH 】 8709, KMUH 19297, KMUH 18702, KMUH 19149, KMUH 18886, KMUH 16758, KMUH 17123 » KMUH 17529 ' KMUH 17641, KMUH 18352, KMUH 16704, KMUH 17006 > KMUH 15546 * KMUH 14687, KMUH 16793, KMUH 15569, KMUH 14384 * KMUH 14821 » KMUH 15627, KMUH 15874, KMUH 12528, KMUH 13025 , KMUH 13115, KMUH 12961, KMUH 13638, KMUH 13790, KMUH 14288, KMUH 14303, KMUH 14494, KMUH 14685, KMUH 1432, KMUH 14551 > KMUH 14637 » KMUH 14155, KMUH 1469, KMUH 9880, KMUH 11037 > KMUH 12087 * KMUH 1118 BU KMUH 12566, KMUH 12569, KMUH 13295 * KMUH 12088 » KMUH 9798, KMUH 9388, KMUH 9776 , KMUH 8222 » KMUH 8127 » KMUH 6754, KMUH 6828, NCKUH 7290, NCKUH 7161 » NCKUH 7910 » NCKUH 7068, NCKUH 7096, NCKUH 7885, NCKUH 7476, NCKUH 7590, AUH 2 , AUH 3, AUH 4, AUH 5, AUH 7, AUH 丨丨* AUH 12, AUH 15, AUH 16 > AUH 20 * AUH 21 > AUH 22 * AUH 23, AUH 24, AUH 25, AUH 26, AUH 27, AUH 28, AUH 30, AUH 31 2 15 95 •20- 146866.doc 201142038 Table 3_ Strain type reference clinical strain total strain number of Mycobacterium tuberculosis CCUG 20993, CCUG 37454 AUH8, KMUHT13769, KMUHT13928, 17 , NCTC 10269, ATCC KMUHT12727 > KMUHT14817 > KMUHT14298 23003 - KMUH0729-31 - KMUH0903-49 - KMUH0903-51 > KMUH0903-92 > KMUH0903-48, KMUH0729-33, KMUH0903-96 Mycobacterium avium
龜分枝桿菌 偶然分枝桿菌 BCRC 1544 卜 ATCC 19421 ,CCUG282]6,CIP 103963,CIP 103964,CIP 103965 > CIP 103968 ATCC 35752,CCUG 37827 > ATCC 35749 ATCC 6841 > ATCC 19542 ,BCRC 15320,CIP 104595 ,JCM 6387 MB 034,MB249,AUH 17,AUH 18,AUH 19,AUH 33,KMUHT12626,KMUHT12063 ,KMUHT13770,KMUH0729-19, KMUH0903-55 ' KMUH0903-28 MB 041 ' KMUHT12169 > KMUHT12117 > KMUHT11768 ' KMUHT12173 > KMUHT12172 MB 018,KMUHT1U69,KMUH0903_10, KMUH0903-16 > KMUH0903-45 > KMUH0903-9,KMUH0903-7,KMUH0729-17, KMUH0903-43 > KMUH0903-42 ' KMUH0903-56,KMUH0903-57,KMUHT14326, KMUHT13902 ' KMUHT11588 19 9 20 胃分枝桿菌 戈登分枝桿菌Mycobacterium tuberculosis, Mycobacterium tuberculosis BCRC 1544, ATCC 19421, CCUG282] 6, CIP 103963, CIP 103964, CIP 103965 > CIP 103968 ATCC 35752, CCUG 37827 > ATCC 35749 ATCC 6841 > ATCC 19542 , BCRC 15320, CIP 104595 ,JCM 6387 MB 034,MB249,AUH 17,AUH 18,AUH 19,AUH 33,KMUHT12626,KMUHT12063 ,KMUHT13770,KMUH0729-19, KMUH0903-55 ' KMUH0903-28 MB 041 ' KMUHT12169 > KMUHT12117 > KMUHT11768 ' KMUHT12173 > KMUHT12172 MB 018, KMUHT1U69, KMUH0903_10, KMUH0903-16 > KMUH0903-45 > KMUH0903-9, KMUH0903-7, KMUH0729-17, KMUH0903-43 > KMUH0903-42 ' KMUH0903-56, KMUH0903-57, KMUHT14326 , KMUHT13902 ' KMUHT11588 19 9 20 Mycobacterium vaccae
CCUG 29062 * CCUG 27846 ,ATCC 25157 CCUG 21801 > ATCC 35759 ,CCUG 21805,CCUG 21806 胞内分枝桿菌 CCUG 28005 > CCUG 28000 ,ATCC 3576卜 ATCC 35763 MB 052,AUH 卜 AUH 14,KMUHT13139, KMUHT11696,KMUHT11697, KMUHT13314(0811) > KMUHT12221 ^ KMUHT13314(0818) » KMUH0903-15 > KMUH0729-1,KMUH0903-69,KMUH0729-2 ,KMUH0903-37 MB 084,MB 085,AUH 6,AUH 29,AUH 35 ,KMUH0903-94,KMUH0903-36, KMUHO卯3·95,KMUH0903-60,KMUH0903-98,KMUHO卯3-90,KMUH0903-31, KMUH0729-14 > KMUH0729-16 > KMUH0903-54,KMUH0903-58,KMUH0903-47, KMUH0903-99 > KMUH0903-100 > KMUH0903-27 » KMUH0903-40 » KMUHT12630 » KMUHTI2466 > KMUHT13858 18 28 I46866.doc -21 · 16201142038CCUG 29062 * CCUG 27846, ATCC 25157 CCUG 21801 > ATCC 35759, CCUG 21805, CCUG 21806 Mycobacterium tuberculosis CCUG 28005 > CCUG 28000, ATCC 3576, ATCC 35763 MB 052, AUH AUH 14, KMUHT13139, KMUHT11696, KMUHT11697 KMUHT13314(0811) > KMUHT12221 ^ KMUHT13314(0818) » KMUH0903-15 > KMUH0729-1, KMUH0903-69, KMUH0729-2, KMUH0903-37 MB 084, MB 085, AUH 6, AUH 29, AUH 35, KMUH0903 -94, KMUH0903-36, KMUHO卯3·95, KMUH0903-60, KMUH0903-98, KMUHO卯3-90, KMUH0903-31, KMUH0729-14 > KMUH0729-16 > KMUH0903-54, KMUH0903-58, KMUH0903 -47, KMUH0903-99 > KMUH0903-100 > KMUH0903-27 » KMUH0903-40 » KMUHT12630 » KMUHTI2466 > KMUHT13858 18 28 I46866.doc -21 · 16201142038
堪薩斯分枝桿菌 ATCC 35775,CCUG 27783 MB078,AUH 10,KMUH0903-9卜 KMUH0903-74 » KMUH0903-33 » KMUH0903- CCUG 27785 30 » KMUH0903-4 ' KMUH0903-26 > KMUHT11533 » KMUHT10784 ^ KMUHT14060 蘭分枝桿菌 * KMUHT12152 > KMUHT11418 CCUG 42422,CCUG 42423 ,CCUG 47901 AUH 13 海洋分枝桿菌 ATCC 927,JCM12275, CCUG 32219 » CCUG 32220 ,CCUG 32221,ATCC 25039 AUH 36 » AUH 37 > AUH 38 不產色分枝桿菌 CCUG 28009,ATCC 25143 ,NCTC 10497 MB 059 培力分枝桿菌 CCUG 27976,CCUG 28064 ,ATCC 23049 MB 804,KMUH0903-n,KMUH0903-84 瘰癘分枝桿菌 CCUG 29045 > CCUG 27782 > CCUG 29053 > CCUG 29055 猿分枝桿菌 CCUG 29114,ATCC 25273 ,ATCC 15080 MB 060 恥垢分枝桿菌 ATCC 19420 > BCRC 10708 ,CCUG 21824,JCM 20379 MB 690 斯氏分枝桿菌 CCUG 37675,NCTC 10829 ,NCTC 10830 土地分枝桿菌 CCUG 42429,CCUG 532, ATCC 25268,ATCC 25269 MB 066 潰瘍分枝桿菌 NCTC ]0417,NCTC 10013 ,NCTC 10445,ATCC 25899 蟾蜍分枝桿菌 CCUG 31306,ATCC 19970 ,ATCC 丨 9973 MB 070,AUH9,AUH32 (未鐘定之非結核分枝 KMUH0729-23 * KMUH0903-17 > KMUH0903- 桿菌) 78,KMUH0903-72,KMUH0729-24, KMUH0903-81 » KMUH0903-22 » KMUH0903-85,KMUH0729-15,KMUH0903-25 總菌種 73 121 4 9 4 6 4 4 5 3 5 4 6 10 194 146866.doc -22· 201142038 表4Mycobacterium kansii ATCC 35775, CCUG 27783 MB078, AUH 10, KMUH0903-9 BU KMUH0903-74 » KMUH0903-33 » KMUH0903- CCUG 27785 30 » KMUH0903-4 ' KMUH0903-26 > KMUHT11533 » KMUHT10784 ^ KMUHT14060 * KMUHT12152 > KMUHT11418 CCUG 42422, CCUG 42423, CCUG 47901 AUH 13 Mycobacterium marinum ATCC 927, JCM12275, CCUG 32219 » CCUG 32220, CCUG 32221, ATCC 25039 AUH 36 » AUH 37 > AUH 38 Mycobacterium auxilis CCUG 28009, ATCC 25143, NCTC 10497 MB 059 Mycobacterium phlei CCUG 27976, CCUG 28064, ATCC 23049 MB 804, KMUH0903-n, KMUH0903-84 Mycobacterium phlei CCUG 29045 > CCUG 27782 > CCUG 29053 > CCUG 29055 Mycobacterium phlei CCUG 29114, ATCC 25273, ATCC 15080 MB 060 Mycobacterium smegmatis ATCC 19420 > BCRC 10708 , CCUG 21824, JCM 20379 MB 690 Mycobacterium tuberculosis CCUG 37675, NCTC 10829 , NCTC 10830 Mycobacterium CCUG 42429, CCUG 532, ATCC 25268, ATCC 25269 MB 066 Mycobacterium ulcerans NCTC ]0417, NCTC 10013, NCTC 10445 ATCC 25899 Mycobacterium tuberculosis CCUG 31306, ATCC 19970, ATCC 丨9973 MB 070, AUH9, AUH32 (uncertained non-tuberculous branch KMUH0729-23 * KMUH0903-17 > KMUH0903- bacillus) 78, KMUH0903-72, KMUH0729- 24, KMUH0903-81 » KMUH0903-22 » KMUH0903-85, KMUH0729-15, KMUH0903-25 Total strain 73 121 4 9 4 6 4 4 5 3 5 4 6 10 194 146866.doc -22· 201142038 Table 4
微生物 菌株 總菌株數 未鑑定菌株數 不一致菌株數 Μ agri CCUG 37673 1 1 0 Μ. aichiense CCUG 37672 1 1 0 Μ. alvei CCUG 37585 ] 1 0 Μ. arupense CCUG 39146 1 1 0 Μ. asiaticum MB 031 1 1 0 Μ. brumae CCUG 37586 1 1 0 Μ. celatum CCUG 39185 ] 1 0 Μ. chitae CCUG 39504 1 1 0 Μ. chlorophenolicum BCRC 13726 1 ] 0 Μ chubuense CCUG 37670 1 1 0 Μ. confluent is CCUG 37513 1 1 0 Μ. diernhoferi BCRC 16395 1 1 0 Μ. fallax CCUG 37584 1 1 0 Μ flavescens MB 017 ] 1 0 Μ. gadium CCUG 37515 1 1 0 Μ. genavense MB 691 1 I 0 Μ. goodii CCUG 52054 1 0 1 Μ. haemophilum CCUG 47452,MB 074 2 2 0 Μ. hibermiae CCUG 39184 1 1 0 Μ. hodleri CCUG 38151 1 1 0 Μ. interjectvm MB 786 1 1 0 Μ. intermedium CCUG 37583 1 1 0 Μ mageritense CCUG 37984 1 1 0 Μ malmoense CIP 105775 1 1 0 Μ. neoaurum BCRC 17230 1 1 0 Μ. obuense CCUG 37669 1 I 0 Μ. phlei BCRC 10707 1 1 0 Μ. pulveris CCUG 37668 1 1 0 Μ. rhodesiae CCUG 37664 1 1 0 Μ. senegalense KMUH0729-23 1 ] 0 Μ septicum CCUG 43574 1 1 0 Μ. shimoidei CCUG 37517 1 1 0 Μ. sphagni BCRC 16397 ] ] 0 Μ. thermoresistibile CCUG 28008 1 1 0 Μ. vaccae BCRC 17229 1 1 0 r r τ ι Ο i 146866.doc -23- 201142038 M. vanbaalenii Μ. wolinskyi Aeromonas hydrophila Bacillus cereus BCRC 16820 KMUH0903-17 BCRC 13880 BCRC 10446The number of total strains of microbial strains is not identified. The number of strains is not consistent. gri agri CCUG 37673 1 1 0 Μ. aichiense CCUG 37672 1 1 0 Μ. alvei CCUG 37585 ] 1 0 Μ. arupense CCUG 39146 1 1 0 Μ. asiaticum MB 031 1 1 0 Μ. brumae CCUG 37586 1 1 0 Μ. celatum CCUG 39185 ] 1 0 Μ. chitae CCUG 39504 1 1 0 Μ. chlorophenolicum BCRC 13726 1 ] 0 Μ chubuense CCUG 37670 1 1 0 Μ. confluent is CCUG 37513 1 1 0 Μ Diernhoferi BCRC 16395 1 1 0 Μ. fallax CCUG 37584 1 1 0 Μ flavescens MB 017 ] 1 0 Μ. gadium CCUG 37515 1 1 0 Μ. genavense MB 691 1 I 0 Μ. goodii CCUG 52054 1 0 1 Μ. haemophilum CCUG 47452,MB 074 2 2 0 Μ. hibermiae CCUG 39184 1 1 0 Μ. hodleri CCUG 38151 1 1 0 Μ. interjectvm MB 786 1 1 0 Μ. intermedium CCUG 37583 1 1 0 Μ mageritense CCUG 37984 1 1 0 Μ malmoense CIP 105775 1 1 0 Μ. neoaurum BCRC 17230 1 1 0 Μ. obuense CCUG 37669 1 I 0 Μ. phlei BCRC 10707 1 1 0 Μ. pulveris CCUG 37668 1 1 0 Μ. rhodesiae CCUG 37664 1 1 0 Μ. senegalense KMUH0729-23 1 ] 0 Μ septicum CCUG 43574 1 1 0 Μ. shimoidei CCUG 37517 1 1 0 Μ. sphagni BCRC 16397 ] ] 0 Μ. thermoresistibile CCUG 28008 1 1 0 Μ. vaccae BCRC 17229 1 1 0 rr τ ι Ο i 146866.doc -23 - 201142038 M. vanbaalenii Μ. wolinskyi Aeromonas hydrophila Bacillus cereus BCRC 16820 KMUH0903-17 BCRC 13880 BCRC 10446
Bacteroides fragilis Citrobacter freundii Edwardsiella tarda Enterobacter cloacae Enterococcus faecalis Escherichia coll Fusobacterium nudeatum Gordonia alkanivorans Gordonia amarae Gordonia nitida Gordonia terrae Gordonia westfalica Klebsiella oxytoca Klebsiella pneumoniae Listeria monocytogenes P eptostreptococcus anaerobicus CCUG 4856 BCRC 12291 BCRC 10670 BCRC 10401 ATCC 19433 BCRC 15513 JCM 11024 DSM 44187 BCRC 13728 DSM 44499 BCRC 13382 DSM 44215 BCRC 13985 BCRC 11644 BCRC 15339 CCUG 7835 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Bacteroides fragilis Citrobacter freundii Edwardsiella tarda Enterobacter cloacae Enterococcus faecalis Escherichia coll Fusobacterium nudeatum Gordonia alkanivorans Gordonia amarae Gordonia nitida Gordonia terrae Gordonia westfalica Klebsiella oxytoca Klebsiella pneumoniae Listeria monocytogenes P eptostreptococcus anaerobicus CCUG 4856 BCRC 12291 BCRC 10670 BCRC 10401 ATCC 19433 BCRC 15513 JCM 11024 DSM 44187 BCRC 13728 DSM 44499 BCRC 13382 DSM 44215 BCRC 13985 BCRC 11644 BCRC 15339 CCUG 7835 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Plesiomonas shigelloides Propionibacterium acnes Proteus mirabilis Providencia rettgeri Pseudomonas aeruginosa Serratia marcescens Shigella boydii Shigella.sonnei Staphylococcus aureus Staphylococcus epidermidis Staphylococcus haemolyticus BCRC 14727 CCUG 1794 BCRC 10726 BCRC 13994 BCRC 10944 BCRC 10948 BCRC 15960 BCRC 15966 BCRC 15290 BCRC 15245 BCRC 15239Plesiomonas shigelloides Propionibacterium acnes Proteus mirabilis Providencia rettgeri Pseudomonas aeruginosa Serratia marcescens Shigella boydii Shigella.sonnei Staphylococcus aureus Staphylococcus epidermidis Staphylococcus haemolyticus BCRC 14727 CCUG 1794 BCRC 10726 BCRC 13994 BCRC 10944 BCRC 10948 BCRC 15960 BCRC 15966 BCRC 15290 BCRC 15245 BCRC 15239
Staphylococcus saprophyticus BCRC 13978 Streptococcus pneumoniae ATCC 33400 Streptococcus pyogenes ATCC 14289 Vibrio parahaemolyticus BCRC 10806 Vibrio vulnificus BCRC 13846 Yersinia enterocoliiica BCRC 13999 總數 1 1 1 ] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 71 I 0 1 0 ] 0 ] 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 ] 0 1 0 ] 0 I 0 70 1 146866.doc -24- 201142038 所有的菌株培養均依照各個菌種中心所建議的培養基( 如 BBLtm 7H11 Agar 以及 L0wenstein-Jensen (L-J) agar slants)和培養條件進行次代培養。 DNA 萃取(DNA extraction)Staphylococcus saprophyticus BCRC 13978 Streptococcus pneumoniae ATCC 33400 Streptococcus pyogenes ATCC 14289 Vibrio parahaemolyticus BCRC 10806 Vibrio vulnificus BCRC 13846 Yersinia enterocoliiica BCRC 13999 Total 1 1 1 ] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 71 I 0 1 0 ] 0 ] 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 ] 0 1 0 ] 0 I 0 70 1 146866.doc -24- 201142038 All strains are cultured according to the recommended medium for each species (eg BBLtm 7H11 Agar and L0wenstein-Jensen) (LJ) agar slants) and culture conditions for secondary culture. DNA extraction
菌株DNA萃取方法為煮濟法(boiled method)(Millar等 人,2000,J· Microbiol. Methods. 42 :139-147):在微量離 心管(Eppendorf)中加入滅菌水,取培養基上的菌落懸浮在 滅菌水中,以乾浴槽加熱至100°C,30分鐘後,再用 12,5 00 rpm離心20分鐘,上清液含有細菌DNA,保存於-70°C。 rRNA基因間隔區及gyrB基因之增幅及定序 分別利用弓I 子 spl (5'-ACCTC CTTTC TAAGG AGCAC C-3,,SEQ ID NO. 36)及 sp2 (5'-GATGC TCGCA ACCAC TATCC A-3.,SEQ ID NO. 37)來放大分枝桿菌的rRNA基因 間隔區(Roth 等人 ’ 2000,J, Clin_ Microbiol· 38:1094-1104);引子Gblf (5,-TGGTT AACGC GCTAT CCAC-3,, SEQ ID NO. 38)&Gblr(5,-ACCAACTCTCGTGCCTTAC-3’,SEQ ID NO. 39)用來放大分枝桿菌的幻;γβ基因,引子 均由生工有限公司(MDBio Inc. ’ Taipei,Taiwan)合成。利 用這兩對引子分別以聚合酶連鎖反應進行增幅,引子對 spl及sp2所增幅產物為rRNA基因間隔區。引子對Gblf及 Gblr所增幅的產物為灯以基因的部份序列。以Applied Biosystems 2720 thermal cycler (Applied Biosystems, Taipei,Taiwan)進行PCR ;總體積為50 μΐ的PCR反應液含 t46866.doc -25* 201142038The strain DNA extraction method is a boiled method (Millar et al., 2000, J. Microbiol. Methods. 42: 139-147): sterilized water is added to a microcentrifuge tube (Eppendorf), and colony suspension on the medium is taken. In a sterilized water, the mixture was heated to 100 ° C in a dry bath, and after 30 minutes, it was centrifuged at 12,500 rpm for 20 minutes. The supernatant contained bacterial DNA and stored at -70 °C. The rRNA gene spacer and the gyrB gene were amplified and sequenced using the spl (5'-ACCTC CTTTC TAAGG AGCAC C-3, SEQ ID NO. 36) and sp2 (5'-GATGC TCGCA ACCAC TATCC A-3, respectively). SEQ ID NO. 37) to amplify the rRNA gene spacer of mycobacteria (Roth et al. '2000, J, Clin_Microbiol 38: 1094-1104); primer Gblf (5,-TGGTT AACGC GCTAT CCAC-3, , SEQ ID NO. 38) & Gblr (5, -ACCAACTCTCGTGCCTTAC-3', SEQ ID NO. 39) used to amplify the illusion of mycobacteria; γβ gene, the primers were all produced by MDBio Inc. 'Taipei , Taiwan) synthesis. These two pairs of primers were used to increase the amplification by polymerase chain reaction. The amplified products of the primers on spl and sp2 were rRNA gene spacers. The product of the amplification of the primers on Gblf and Gblr is a partial sequence of the gene. PCR was performed with an Applied Biosystems 2720 thermal cycler (Applied Biosystems, Taipei, Taiwan); a total volume of 50 μM PCR reaction solution containing t46866.doc -25* 201142038
有 DNA模板(約 50 ng),75 mM Tris-HCl (pH 8.5),20 mM ammonium sulfate , 1.5 mM MgCl〗,0.8 mM deoxyribonucleoside triphosphates (0.2 mM each) ’ 1 M (each) primer,及 1.25 U of GoTaq® Hot Start polymerase (Promega,Madison,WI,USA)。PCR反應為初始變性 (initial denaturation,94°C,3 mins),40個循環的變性DNA template (approximately 50 ng), 75 mM Tris-HCl (pH 8.5), 20 mM ammonium sulfate, 1.5 mM MgCl, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each) ' 1 M (each) primer, and 1.25 U of GoTaq® Hot Start polymerase (Promega, Madison, WI, USA). The PCR reaction was initial denaturation (94 ° C, 3 mins), 40 cycles of denaturation
(denaturation,94°C,1 min),黏合(annealing,59°C,1 min)和延伸(extension,72°C,1_5 mins)反應,最後為 7分 鐘的延伸反應(extension,72°C,7 mins) (Gurtler ei α/· ’ 2006)分別增幅產物之後,以2%的瓊脂(agarose, AMRESCO®,OH,USA)進行電泳產物分析。若PCR產物 要和晶片進行雜合反應時,則各引子對[(spl,sp2)及 (Gblf,Gblr)]中之一引子或二引子之5’端標記毛地黃素。 特異性探針(specific probes)之設計 將參考菌株定序,加上GenBank資料庫上大量搜集分枝 桿菌的rRNA基因間隔區及基因序列,利用序列比對 軟體 Vector NTI (Invitrogen Corporation,Carlsbad,CA, USA)進行序列比對(sequence alignment)之後,找出種與種 之間(interspecies)的差異性序列,以及同種不同菌株間 (intraspecies)的相似性序列來設計探針。本研究之晶片共 有32個探針,包含1個分枝桿菌正對照組探針 (mycobacteria positive control,代號為PC)及 13 個座標標 記探針(position maker ’代號M)以及3 1個菌種特異性探針( 圖1)。正對照組探針的選定採用盡可能蒐集到的分枝桿菌 146866.doc -26- 201142038 rRNA基因間隔區中的高度保留區域(c〇nserve(j regi〇n);座 標標記探針選用已標記毛地黃素之沒有菌種特異性之募核 苷酸探針。 以製造點突變(Site direct mutation)方式產生模擬PCR產物 在MTBC中’極罕見的菌種坎結核桿 菌(M. 在全 世界的菌種中心均沒有供應,故以人工合成的坎結核桿菌 灯Μ基因的序列,來測試探針是否能鑑定坎結核桿菌。從 GenBank收集到的MTBC菌種中發現,坎結核桿菌和結核 桿菌之幻基因序列間僅有單一個核苷酸差異(SNp),故 選殖結核桿菌H37Rv的幻^片段,再利用QuikChange® Π site-directed mutagenesis kit (Stratagene Products > Hwy > USA) ’在結核桿菌H37Rv菌株的g少序列上製造一個跟坎 結核桿菌相同的單一個核苷酸差異,即可模擬坎結核桿菌 之PCR產物,經定序確認正確無誤後,以套組 (Geneaid plasmid midi kit,Taiwan)抽取細菌質體,收集模 擬的片段並保存於-80°C,以供日後使用。由於MTBC中所 有菌種rRNA基因間隔區序列的相似度為i 〇〇%,在測試坎 結核桿菌時,是以模擬之坎結核桿菌和結核桿菌 H37Rv之rRNA基因間隔區的PCR產物進行雜合反應。 分枝桿菌晶片製備 分別將探針與染劑(tracking dye)以體積比1:1 (vol/vol) 混合於96孔圓底的微量盤(ELISA plate,Becton Drive,NJ ’ USA) ’探針最終濃度除正負對照組及座標探針之外, 其他皆為1 〇 μΜ ;正對照組(positive control,PC)濃度為5 146866.doc -27- 201142038 μΜ,座標探針濃度為2.5 μΜ,負對照組(negative control ,NC)則疋由無菌水和染劑等體積混合而成。 將配置完成的微量盤置於EZSp〇tTM arrayer SR-A300 (悅 生科技’台北’台灣)’利用直徑為400 μηι的實心針頭 ( Ρ·η)把各個揼針點在尼龍膜(positively charged(denaturation, 94 ° C, 1 min), adhesion (annealing, 59 ° C, 1 min) and extension (extension, 72 ° C, 1_5 mins) reaction, and finally a 7-minute extension reaction (extension, 72 ° C, After 7 mins) (Gurtler ei α/· ' 2006), the products were analyzed by electrophoresis with 2% agar (agarose, AMRESCO®, OH, USA). If the PCR product is to be heterozygously reacted with the wafer, the primers are labeled with digoxigenin at the 5' end of one of the primers or the two primers of [(spl, sp2) and (Gblf, Gblr)]. The design of specific probes will be sequenced with reference strains, and a large number of rRNA gene spacers and gene sequences of mycobacteria collected on the GenBank database, using sequence alignment software Vector NTI (Invitrogen Corporation, Carlsbad, CA) , USA) After performing sequence alignment, find the differential sequences between species and interspecies, and sequence similar sequences of different intraspecies to design probes. The wafers in this study consisted of 32 probes, including one mycobacteria positive control (code-named PC) and 13 coordinate-labeled probes (position maker 'code M) and 31 strains. Specific probe (Figure 1). The positive control group was selected using the as much as possible collection of the mycobacteria 146866.doc -26- 201142038 rRNA gene spacer in the highly reserved region (c〇nserve (j regi〇n); coordinate marker probe selected marked There is no strain-specific nucleotide probe for foxglovein. Producing a simulated PCR product in the form of a site mutation, a very rare strain of M. tuberculosis (M. in the world) There is no supply at the center, so the sequence of the synthetic tuberculosis gene of the tuberculosis can be used to test whether the probe can identify Mycobacterium tuberculosis. The MTBC strain collected from GenBank found that the tuberculosis of Mycobacterium tuberculosis and Mycobacterium tuberculosis There is only one single nucleotide difference (SNp) between the gene sequences, so the genomic fragment of Mycobacterium tuberculosis H37Rv is selected, and then QuikChange® Π site-directed mutagenesis kit (Stratagene Products > Hwy > USA) 'in Mycobacterium tuberculosis The H1Rv strain has a single nucleotide difference in the same sequence of G. tuberculosis, and the PCR product of M. tuberculosis can be simulated. After confirming the correctness by sequencing, The bacterial plastids were collected from the Geneaid plasmid midi kit (Taiwan), and the mock fragments were collected and stored at -80 ° C for later use. Since the similarity of the rRNA gene spacer sequences of all strains in MTBC is i 〇〇 %, in the test of Mycobacterium tuberculosis, the heterozygous reaction was carried out by PCR products of the rRNA gene spacer of M. tuberculosis and Mycobacterium tuberculosis H37Rv. Mycobacterial wafer preparation separately used probes and tracking dyes Volume ratio 1:1 (vol/vol) mixed in a 96-well round bottom microplate (ELISA plate, Becton Drive, NJ 'USA) 'The final concentration of the probe except the positive and negative control group and the coordinate probe, all other 1 〇μΜ; positive control (PC) concentration is 5 146866.doc -27- 201142038 μΜ, coordinate probe concentration is 2.5 μΜ, negative control (negative control, NC) is from sterile water and dye, etc. The volume is mixed. Place the configured microplate in the EZSp〇tTM arrayer SR-A300 (Yuesheng Technology 'Taipei' Taiwan') using a solid needle ((·η) with a diameter of 400 μηι. Positive charge d
Nylon membrane,Roche ’ Mannheim,Germany)上,二點 之間的中心距離為8〇〇 μηι。此晶片上共有49點χ 7 d〇ts) ’大小為6.8 mm x 6_8 mm。晶片製作完成後,以On the Nylon membrane, Roche ’ Mannheim, Germany, the center distance between the two points is 8〇〇 μηι. There are 49 points χ 7 d〇ts) on this wafer. The size is 6.8 mm x 6_8 mm. After the wafer is finished,
shortwave UV (Stratalinker 1800; Stratagen,La Jolla,CA ’ USA) 3 W/cm2能量之紫外光照射3〇秒,促使探針固定在 尼龍膜上,接著儲存在陰暗乾燥處備用。微陣列晶片探針 位置示於圖1。 刀枝杯_日曰片的雜合反應hybridizcition) 於製作好的分枝桿菌晶片甲,分別加入已標記毛地黃素 的PCR產物’進行雜合反應。雜合反應進行的主要步驟如 下: 將製作好的晶片標記之後,利用0.5X的SSC緩衝液 [lx SSC 緩衝液為 0.15 M NaCl 和 0·015 M sodium citrate)-〇.-i % sodium dodecyl sulfate (SDS ,Shortwave UV (Stratalinker 1800; Stratagen, La Jolla, CA' USA) 3 W/cm2 of UV light was irradiated for 3 sec seconds, causing the probe to be attached to a nylon membrane and then stored in a dark dry place for later use. The microarray wafer probe locations are shown in Figure 1. Hybridizcition of the stalk cup _ 曰 曰 ) ) 于 制作 hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy hy The main steps of the hybridization reaction are as follows: After labeling the prepared wafer, use 0.5X SSC buffer [lx SSC buffer is 0.15 M NaCl and 0·015 M sodium citrate)-〇.-i % sodium dodecyl sulfate (SDS,
Boehringer Mannheim,Mannheim,Germany)]清洗 晶片’以震盪器震盪4次,每次3分鐘,以洗去晶片 上的追蹤染劑、未結合在尼龍膜上的探針及其他雜 質 2.加入雜合緩衝液[hybridization solution,5X SSC, 146866.doc -28- 201142038 1% (w/v) blocking reagent (Roche Diagnostics, Indianapolis ’ IN ’ USA),0.1% N-laurylsarcosine and 0.02% SDS],在室溫下震盪2小時進行前雜合反 應(prehybridization) 3. 把每一晶片依序放入24孔的細胞培養盤(24-well cell culture plate)每一孔中,每孔加入300 μΐ的雜合緩衝 液 4. 將帶有毛地黃素的PCR產物加熱至95°C,5分鐘,用 • 以解旋(denature)成單股狀態,並立刻置於冰上 5. 在冰浴上’分別將各個單股狀態的PCR產物取10 μΐ 加入含有雜合緩衝液的晶片中,在雜合反應箱 (hybridization oven ’ Firstek Scientific,Taipei, Taiwan)中(50°C,60rpm)進行1.5小時的雜合反應 6_用0.25 x SSC緩衝液作為清洗緩衝液(wash buffer), 清洗4次’每次5分鐘,洗掉未結合的PCR產物和雜 合緩衝液 7·移除清洗緩衝液,加入lx填塞緩衝液[blocking buffer,10% blocking reagent (Roche Diagnostics)以 maleic acid buffer (0.1 M maleic acid,0.15 M NaCl ,pH 7.5)稀釋10倍],室溫震盪i小時 8· 加入稀釋2500倍(以1 X blocking buffer稀釋)標記有 驗性填酸酶(alkaline phosphatase,AP)之抗毛地黃 素抗體(anti-digoxigenin-AP Fab fragments,Roche) ,室溫震盪.反應1小時 146866.doc •29· 201142038 9.移除抗體緩衝液’接著以緩衝液清洗[〇 3% (v/v) Tween 20 in ΐχ maieic acid buffer]洗去未結合的抗 體,室溫下清洗3次,每次1 5分鐘 1 〇·加入偵測緩衝液(detection buffer,0· 1 M Tris-HCl, 〇·15 M NaCl ’ pH 9.5)室溫震盪5分鐘後,去除偵測 緩衝液 11. 以倘測緩衝液將鹼性磷酸酶受質[st〇ck s〇luti(m 〇f mtroblue tetrazolium chloride/5-bromo-4-chloro-3 -indolylphosphate (NBT/ BCIP),R0Che]稀釋 50倍, 每個晶片上加40 μΐ,37°C避光,進行25分鐘至45分 鐘的呈色反應,反應進行中不要搖晃。 12. 用純水清洗晶片4次,每次5分鐘,以洗掉NBT/BcIp 終止呈色反應 13,放入烘箱中烘乾後,即可用肉眼判讀結果 晶片雜合反應之結果判讀 B曰片上有座標標記,可辨別上下左右,座標標記將晶片Boehringer Mannheim, Mannheim, Germany)] The cleaning wafer was oscillated 4 times with an oscillator for 3 minutes each time to wash away trace dye on the wafer, probes not bonded to the nylon membrane and other impurities. Buffer [hybridization solution, 5X SSC, 146866.doc -28- 201142038 1% (w/v) blocking reagent (Roche Diagnostics, Indianapolis ' IN 'USA), 0.1% N-laurylsarcosine and 0.02% SDS], at room temperature Prehybridization for 2 hours under shaking 3. Place each wafer sequentially into each well of a 24-well cell culture plate, and add 300 μΐ of hybrid buffer to each well. Liquid 4. Heat the PCR product with digoxigenin to 95 ° C for 5 minutes, use • to de-spin into a single-strand state, and immediately place on ice. 5. On the ice bath, respectively 10 μΐ of the PCR product of each single-stranded state was added to the wafer containing the hybrid buffer, and hybridization was carried out for 1.5 hours in a hybridization oven 'Firstek Scientific, Taipei, Taiwan (50 ° C, 60 rpm). Reaction 6_ with 0.25 x SSC buffer Wash buffer (wash buffer), wash 4 times '5 minutes each time, wash away unbound PCR product and hybrid buffer 7 · Remove wash buffer, add lx stuffing buffer [blocking buffer, 10% blocking reagent (Roche Diagnostics) diluted 10 times with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5), shaking at room temperature for 1 hour 8 · adding 2500 times dilution (diluted with 1 X blocking buffer) Acidase phosphatase (AP) anti-digoxigenin-AP Fab fragments (Roche), shaking at room temperature. Reaction 1 hour 146866.doc •29· 201142038 9. Remove antibody buffer' Wash the unbound antibody by buffer [〇3% (v/v) Tween 20 in ΐχ maieic acid buffer], and wash it 3 times at room temperature for 1 5 minutes 1 〇·Add detection buffer (detection Buffer, 0· 1 M Tris-HCl, 〇·15 M NaCl 'pH 9.5) After shaking for 5 minutes at room temperature, remove the detection buffer 11. If the assay buffer will alkaline phosphatase [st〇ck s 〇luti(m 〇f mtroblue tetrazolium chloride/5-bromo-4-chloro-3 -indolylp Hosphate (NBT/ BCIP), R0Che] diluted 50 times, add 40 μΐ to each wafer, and avoid light at 37 ° C for 25 minutes to 45 minutes. Do not shake during the reaction. 12. Clean the wafer 4 times with pure water for 5 minutes each time to wash off the NBT/BcIp to terminate the color reaction 13. After drying in an oven, the results of the hybrid reaction of the wafer can be judged by the naked eye. There are coordinate marks to identify the top, bottom, left and right, and the coordinate mark will be wafer
刀^成兩大區域。晶片上半部區域的探針用來鑑定MTBC 八内不同鹵種,及兩個負對照組(negative⑶ntr〇丨,nc) ;下半部區域的探針用來鑑定NTM,及一個正對照組 (positive _r。卜Pc) #針,該探針可以和分枝桿菌屬細 菌反應。 晶片反應結果必須^^0點沒有雜合反應發生,而PC點有 雜σ反應才旎進行結果判讀。若鑑定mtbc的二個探針均 有反應,表示檢體中有肘1^(:存在,至於是肘丁8(:中的那 146866.doc •30- 201142038 一種’將於下文中詳細說明。NTM的鑑定則看是那一個 NTM的探針發生雜合反應。 rRNA基因間隔區,16S rRNA基因及gyrB之定序 以晶片鑑定結果若種名和菌種中心的種名不符合,或和 傳統(或其他分生方法)所鑑定之菌名不一致時,則定序此 菌種之rRNA基因間隔區、16S rRNA基因、或序列, 作為第三種參考方法。純化之PCR產物進行定序,使用核 酉义疋序儀 PRISM 3100 Sequencer (Applied Biosystems, • Taipei ’ Taiwan) ’ 及 BigDye Terminator cycle sequencing kit (version 3.7; Applied Biosystems)定序。 靈敏度、特異性以及偵測極限之定義 靈敏度(sensitivity):欲鑑定的目標菌株得到正確鑑定的 百为比[sensitivity = (no. of target strains correctly identified/total no. of target strains tested) χ 100%] 特異性(specificity):非目標菌株,沒有得到鑑定的百分 比[specificity = (no. of nontarget strains not 9 identified/total no. of nontarget strains tested) χ 100%] 偵測極限(detection limit):待測菌株能被晶片檢測到的 最低genomic DNA量。Knife ^ into two major areas. Probes in the upper half of the wafer were used to identify different halogen species in MTBC, and two negative control groups (negative(3)ntr〇丨, nc); probes in the lower half were used to identify NTM, and a positive control group ( Positive _r. Pc) #针, the probe can react with mycobacteria bacteria. The result of the wafer reaction must have no heterozygous reaction at the ^^0 point, and the PC point has a heterozygous sigma reaction before the result is interpreted. If both probes for the identification of mtbc are reactive, it indicates that there is elbow in the specimen (: exists, as for the elbow 8 (the 146866.doc • 30- 201142038 in a 'will be described in detail below). The identification of NTM is based on the heterozygous reaction of the NTM probe. The rRNA gene spacer, the 16S rRNA gene and the gyrB sequence are identified by the wafer if the species name does not match the species name of the center of the strain, or Or if the bacterial names identified by other sub-methods are inconsistent, the rRNA gene spacer, 16S rRNA gene, or sequence of the strain is sequenced as a third reference method. The purified PCR product is sequenced and used. The sequencing of the PRISM 3100 Sequencer (Applied Biosystems, • Taipei 'Taiwan)' and the BigDye Terminator cycle sequencing kit (version 3.7; Applied Biosystems). Sensitivity of Sensitivity, Specificity, and Detection Limits: To be identified The target strain is correctly identified. [sensitivity = (no. of target strains correctly identified/total no. of target strains tested) χ 100%] specificity (s Pecificity): non-target strain, % not identified [specificity = (no. of nontarget strains not 9 identified/total no. of nontarget strains tested) χ 100%] detection limit: the test strain can be The minimum amount of genomic DNA detected by the wafer.
Mycobacterium spp.正對照組(positive c〇ntr〇i,ρ〇 探針 此探針為分枝桿菌菌屬探針,由分枝桿菌屬 spp.)之rRNA基因間隔區序列中的共同序 列所設計,探針代號為Myc2及Myc3,由於探針Myc2無法 ' 涵蓋所有的分枝桿菌,針對無法涵蓋的菌種另設計Myc3 I46866.doc 31 .201142038 ’再將兩者混合在一起,作 pc。本研究巾,當此探針發 分枝桿菌屬細菌。 為分枝桿菌菌屬探針’代號為 生雜合反應,表示待測菌種為 結核桿菌群(MTBC)之鐘定 ’。核知菌群的特異性探針共分為兩部份一部分是針對 MTBC所設計的菌雜$太田Mycobacterium spp. is a control group (positive c〇ntr〇i, ρ〇 probe is a probe of Mycobacterium spp., which is a common sequence in the rRNA gene spacer sequence of Mycobacterium spp.) The probes are coded as Myc2 and Myc3. Since the probe Myc2 cannot 'cover all mycobacteria, design Myc3 I46866.doc 31 .201142038' for the species that cannot be covered, and then mix the two together for pc. This study towel, when this probe is a Mycobacterium bacterium. For the Mycobacterium sp. probe, the code is a heterozygous reaction, indicating that the test species is the bell bore of the Mycobacterium tuberculosis group (MTBC). The specific probe of the known bacterial group is divided into two parts, which are designed for the MTBC.
園群特異性探針,另一部分是針對MTBC 中的各個菌種設計的菌種探針。The cluster-specific probes, and the other part are strain probes designed for each species in MTBC.
MTBC菌群的探針有兩個,代冑分別為MTBC2和MTBC4 探針長度分別為18個及19個核苦酸,由⑽此的:⑽八基 因間隔區上共有的序列所設計其序列與ntm菌種有很大 的差異’凡屬於結核桿菌群的㈣,包括非洲結核桿菌, 牛、’σ核杯菌群,坎結核桿菌,卡柏結核桿菌,邁克結核桿 菌’和結核才旱菌均會和在匕二探針產生雜合反應;結核桿菌 群以外的g種’皆不會和此二探針產生反應。 MTBC菌種的特異性探針共有12個,探針代號MtublR及 MtubWIR用來鑑定結核桿菌,若菌株為結核桿菌則 MtublR 的雜合訊號會比 MtubwlR 強;Mb〇vG1&Mb〇vGwl 用來鑑定牛結核桿菌群,包括牛結核桿菌、牛結核桿菌 BCG、及卡柏結核桿菌和Mb〇vG1探針的雜合訊號比和 MbovGW 1的雜合訊號強,但無法區分出牛結核桿菌群内 的菌種。Mbovl及MbovW1用來鑑定牛結核桿菌,包括牛 結核桿菌以及牛結核桿菌BCG* Mb〇vl的雜合訊號都比和 Mb〇vW1的雜合訊號強;Mcapl及McapWl為卡柏結核桿菌 的探針,僅卡柏結核桿菌才會呈現Mcapl比McapW1強的 146866.doc •32· 201142038 雜合訊號;Mmicl及MmicWl為邁克結核桿菌的探針,僅 邁克結核桿菌才會呈現Mmicl比MmicWl強的雜合訊號; Mcanl及McanWl為坎結核桿菌的探針,由於坎結核桿菌 和結核桿菌在幻基因上序列較相似,故坎結核桿菌的雜 合反應會呈現MtublR比MtubWIR強的雜合訊號,以及 Mcanl比McanWl強的反應訊號。 MTBC菌種的鑑定,首先需要菌群探針Mtbc2及MTBC4 同時有反應的情況下(即待測菌種屬於MTBC),才進—步 鑑定是該群内的那一菌種,以雜合圖譜型式(hybridizati〇n pattern)做為囷種鑑疋依據。以結核桿菌為例,它的圖譜 型式為:MTBC2及MTBC4同時有反應,MtublIUf針的反 應比MtubWl R強以外,且剩下1 〇個鑑定mtbC中菌種的探 針,均同時證明其不是MTBC中的其他菌種(即探針代號中 有”W”字母的探針,其反應比同—組中代號沒有” W”的探 針反應強)。卡柏結核桿菌以前被放在牛結核桿菌群内, 現被命名為卡柏結核桿菌(Aranaz等人,2003,j Syst Evol. Microbi〇i. 53:1 785_1789),它的探針反應圖譜為 MbovGl比MbovGWl反應強(即屬於牛結核桿菌群),且 Mcapl比McapW1反應強(即它是卡柏結核桿菌)。 非結核分枝桿菌(NTM)之鑑定 針對非結核分枝桿菌所設計之菌種探針共有17個,其中 有兩個振針為群探針(complex probe),共可鑑定19種1^丁^1 目標菌種,分別為潰瘍腫分枝桿菌、鳥分枝桿菌、龜分枝 桿菌、偶然分枝桿菌、胃分枝桿菌、戈登分枝桿菌、胞内 146866.doc -33- 201142038 分枝桿菌'堪薩斯分枝桿菌、蘭分枝桿菌 、:f色分枝桿菌、培力分枝桿菌、痛癘分枝桿;= 枝杯囷、恥垢分枝桿菌、斯 义刀 、、眚瘟八妯垆# 八刀枝才干痛、土地分枝桿菌、 ^廣刀枝杯函、及蟾蜍分枝桿菌,其中Μηι_2 海洋分枝桿菌或潰瘍分枝桿g, m心 桿菌或蘭分枝桿菌。 —來鐘定猿分枝There are two probes for the MTBC flora, which are 18 and 19 nucleotides of MTBC2 and MTBC4, respectively. The sequence is designed by (10) the sequence shared by the (10) octal spacer. There are great differences in ntm strains. (4) Those belonging to the Mycobacterium tuberculosis group, including Mycobacterium tuberculosis, cattle, 'σ nucleocapsid group, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis' and Mycobacterium tuberculosis It will produce a heterozygous reaction with the second probe; the g species other than the Mycobacterium tuberculosis will not react with the two probes. There are 12 specific probes for MTBC strains. The probe codes MtublR and MtubWIR are used to identify Mycobacterium tuberculosis. If the strain is Mycobacterium tuberculosis, the hybrid signal of MtublR will be stronger than MtubwlR; Mb〇vG1&Mb〇vGwl is used to identify The Mycobacterium tuberculosis group, including Mycobacterium tuberculosis, Mycobacterium tuberculosis BCG, and the K. tuberculosis and Mb〇vG1 probes, has a heterozygous signal ratio and MbovGW 1 heterozygous signal, but cannot distinguish between the M. tuberculosis population. Species. Mbovl and MbovW1 are used to identify Mycobacterium bovis, including Mycobacterium tuberculosis and Mycobacterium tuberculosis BCG* Mb〇vl. The hybrid signal is stronger than the hybrid signal of Mb〇vW1; Mcapl and McapWl are probes of Mycobacterium tuberculosis Only Kappa tuberculosis will present Mcapl stronger than McapW1 146866.doc •32· 201142038 Hybrid signal; Mmicl and MmicWl are probes of M. tuberculosis, only M. tuberculosis will present Mmicl heterozygous than MmicWl Mcanl and McanWl are probes of Mycobacterium tuberculosis. Because the sequence of Mycobacterium tuberculosis and Mycobacterium tuberculosis are similar in the phantom gene, the heterozygous reaction of Mycobacterium tuberculosis will show a strong hybrid signal of MtublR than MtubWIR, and the ratio of Mcanl. McanWl strong response signal. The identification of MTBC strains first requires the microbial probes Mtbc2 and MTBC4 to react at the same time (ie, the tested species belong to MTBC), and then the identification is the one in the group, with a heterozygous map. The type (hybridizati〇n pattern) is used as a basis for consideration. Take Mycobacterium tuberculosis as an example, its pattern type is: MTBC2 and MTBC4 react at the same time, MtublIUf needle reaction is stronger than MtubWl R, and there are 1 probes for identifying mtbC strains, which also prove that it is not MTBC. The other species in the strain (ie, the probe with the "W" letter in the probe code reacted more strongly than the probe in the same group with no "W" code). K. tuberculosis was previously placed in the Bovine tuberculosis group and is now named K. tuberculosis (Aranaz et al., 2003, j Syst Evol. Microbi〇i. 53:1 785_1789). Its probe response profile is MbovGl reacts strongly with MbovGW1 (ie belongs to the population of Mycobacterium bovis), and Mcapl reacts more strongly than McapW1 (ie it is K. tuberculosis). Identification of non-tuberculous mycobacteria (NTM) There are 17 species of probes designed for non-tuberculous mycobacteria. Two of the vibrating needles are complex probes, and a total of 19 species can be identified. ^1 Target strains, respectively, Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium vaccae, Mycobacterium gordonii, intracellular 146866.doc -33- 201142038 Mycobacterium 'M. kansii, Mycobacterium marinum, : Mycobacterium f, Mycobacterium phlei, Mycosis branch; = Twig, M. smegmatis, Syzygium, 眚瘟Gossip #八刀枝才干痛, Mycobacterium phlei, ^Guangdaozhi cup, and Mycobacterium sputum, of which Μηι_2 marine mycobacteria or ulcer branching rod g, M. aureus or Mycobacterium lanatus. - Come to Zhongding Branch
雜合反應結果必須正對照組(PC)有反應,接著看那一探 ^有反應’例如PC和Mehe3二個探針有反應,菌株鑑定為 龜为枝桿菌。唯—例外是胞内分枝桿菌會㈣定鳥分枝桿 因的探針(MaVll)有輕微交又反應,所以Mint2 (鑑定胞内 ^枝^的探針)#aMavil同時有反應時,菌株鐘定為胞内 刀枝才干菌,但Mint2的反應較]^…強很乡。若Pc及m 有雜合反應則鑑定為鳥分枝㈣,這是由於鳥分枝桿菌和 胞内分枝桿g同屬於烏分枝桿菌群⑽_㈣請 C〇mpleX) ’兩者在生化反應及rRNA基因間隔區序列上非常 相似。臨床上不易區分出二菌種’通常只鑑定到鳥分枝桿 菌群。 芬考逢株之晶片測試 的片的測试結果如圖2及圖3所示,共測試了 88株參考菌 株和1個幻^方模擬序列,包括MTBC 15株及NTM 73株。另 測試目標臨床菌株,其中MTBC共8〇株(表2),NTM 121株 ,共201株(表3)。 为枝桿菌晶片之靈敏度(sensitivity)與特異性(speci^icity) 所有目標菌株289株,其中有二株(μ.々α謂j/z· I46866.doc -34- 201142038 KMUH0903-4 及 M. h«iflfi/zKMUH0903-26)晶片鑑定錯誤 ,所以晶片靈敏度為99.3% (287/289)(表2和表3)。所測試 的非目.標菌(nontarget species) 73株中(表4),其中有一株 鑑定錯誤(Λ/· CCUG 52054被鑑定為, 所以晶片特異性為98.6% (72/73)。 晶片之 <貞測極限(detection limit) 以結核桿菌H37Rv,潰瘍腫分枝桿菌CCUG 20993τ和 偶然分枝桿菌JCM 6387作為測試菌株。DNA濃度均由10 ng/μΐ做10倍序列稀釋。結核桿菌H37Rv的偵測極限為10 fg/μΐ (即50 fg/每一測試),約相當於12個細菌的DNA含量 [(一個 £_ Co"細胞含有 4 fg DNA (Kubitschek and Friedman ’ 1971)]。潰瘍腫分枝桿菌CCUG 20993T和偶然分枝桿菌 】〇]\4 63 87的檢測極限均為1〇〇『§41(即5〇0[§/每一測試), 約相當於120個細菌的DNA含量。 上述實施例僅為說明本發明之原理及其功效,並非限制 本發明。因此習於此技術之人士對上述實施例進行修改及 變化仍不脫本發明之精神。本發明之權利範圍應如後述之 申請專利範圍所列。 【圖式簡單說明】 圖1顯示本發明微陣列晶片探針位置示意圖。 圖2顯示結核桿菌群之目標菌之雜合反應圖: (1) 非洲結核桿菌ATCC 25420 (2) 非洲結核桿菌ATCC 3571 1 (3) 牛結核桿菌ATCC 27291 146866.doc •35- 201142038 (4) 牛結核桿菌ATCC 35734 (5) 牛結核桿菌BCG ATCC 3573 1 (6) 牛結核桿菌BCG ATCC 35735 (7) 牛結核桿菌BCG ATCC 35737 (8) 卡柏結核桿菌ATCC BAA-824 (9) 坎結核桿菌 (10) 邁克結核桿菌ATCC 19422 (11) 邁克結核桿菌ATCC 35782 • (12)邁克結核桿菌NCTC 8337 (13) 結核桿菌H37Rv (14) 結核桿菌 ATCC 25177 (15) 結核桿菌 ATCC 358 1 8 (16) 結核桿菌 ATCC 35828 圖3顯示非結核分枝桿菌目標菌(1-1 9)與非目標菌(20-32) 之雜合反應圖 (1)潰瘍腫結核桿菌CCUG 20993 ® (2)鳥分枝桿菌BCRC 15441 (3) 龜分枝桿菌CCUG 35749 (4) 偶然分枝桿菌BCRC 1 5320 (5) 胃分枝桿菌CCUG 29062 (6) 戈登分枝桿菌CCUG 21801 (7) 胞内分枝桿菌CCUG 28005 (8) 堪薩斯分枝桿菌CCUG 27785 (9) 蘭分枝桿菌CCUG 47901 I46866.doc -36· 201142038 (10) 海洋分枝桿菌ATCC 25039 (11) 不產色分枝桿菌CCUG 28009 (12) 培力分枝桿菌CCUG 27976 (13) 瘰癘分枝桿菌CCUG 29045 (14) 猿分枝桿菌CCUG 29114 (15) 恥垢分枝桿菌ATCC 19420 (16) 斯氏分枝桿菌NCTC 10829 (17) 土地分枝桿菌CCUG 42429 • (18)潰瘍分枝桿菌ATCC 25899 (19) 蟾蜍分枝桿菌ATCC 19973 (20) 分枝桿菌 Μ CCUG 37673 (21) 分枝桿菌 Μ. α/vez· CCUG 3 7585 (22) 分枝桿菌 M CCUG 39146 (23) 分枝桿菌 M. asiaticum MB 03 1 (24) 分枝桿菌 M. e/nke CCUG 39504 (25) 分枝桿菌 M. c/z/or<9/?/ze«o/z_cwwBCRC 13726The results of the heterozygous reaction must be positive in the control group (PC), and then the reaction is observed. For example, two probes, PC and Mehe3, react, and the strain is identified as Mycobacterium. The only exception is that the intracellular mycobacteria will (4) the probe of the bird's branching rod (MaVll) has a slight crossover reaction, so Mint2 (the probe that identifies the intracellular ^ branch ^) #aMavil also reacts when the strain Zhong Ding is an intracellular bacterium, but the reaction of Mint2 is stronger than that of ^^. If there is a heterozygous reaction between Pc and m, it is identified as a bird branch (4). This is because Mycobacterium avium and intracellular branching rod g belong to the mycobacterium group (10)_(4) Please C〇mpleX) 'both in biochemical reaction and The rRNA gene spacer sequences are very similar. It is not easy to distinguish the two strains clinically. Usually only the avian flora is identified. The results of the test of the Fen Kao's wafer test are shown in Figure 2 and Figure 3. A total of 88 reference strains and one mimetic simulation sequence were tested, including MTBC 15 strain and NTM 73 strain. The target clinical strains were also tested, including 8 BC strains (Table 2) and NTM 121 strains (201) (Table 3). Sensitivity and specificity (speci^icity) of all the target strains of 289 strains, of which there are two strains (μ.々α, j/z· I46866.doc -34- 201142038 KMUH0903-4 and M. h«iflfi/zKMUH0903-26) Wafer identification error, so the wafer sensitivity was 99.3% (287/289) (Table 2 and Table 3). Among the 73 strains of nontarget species tested (Table 4), one of them was identified incorrectly (Λ/· CCUG 52054 was identified as, so the wafer specificity was 98.6% (72/73). <detection limit As the test strain, Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis CCUG 20993τ and Mycobacterium tuberculosis JCM 6387 were used as test strains. The DNA concentration was 10 times serial dilution by 10 ng/μΐ. Mycobacterium tuberculosis H37Rv The detection limit is 10 fg/μΐ (ie 50 fg/per test), which is equivalent to the DNA content of 12 bacteria [(a £_Co" cells contain 4 fg DNA (Kubitschek and Friedman ' 1971)]. The detection limit of Mycobacterium CCUG 20993T and Occasional Mycobacterium 〇]\4 63 87 is 1〇〇“§41 (ie 5〇0[§/each test), which is equivalent to the DNA content of 120 bacteria. The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. The modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the scope of the invention. The scope of the patent application mentioned later is listed. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the position of a probe of a microarray wafer of the present invention. Fig. 2 is a diagram showing the hybrid reaction of a target strain of Mycobacterium tuberculosis: (1) Mycobacterium tuberculosis ATCC 25420 (2) Mycobacterium tuberculosis ATCC 3571 1 (3) Bovine tuberculosis ATCC 27291 146866.doc •35- 201142038 (4) Bovine tuberculosis ATCC 35734 (5) Bovine tuberculosis BCG ATCC 3573 1 (6) Bovine tuberculosis BCG ATCC 35735 (7) Bovine tuberculosis BCG ATCC 35737 ( 8) Mycobacterium tuberculosis ATCC BAA-824 (9) Mycobacterium tuberculosis (10) Mycobacterium tuberculosis ATCC 19422 (11) Mycobacterium tuberculosis ATCC 35782 • (12) Mycobacterium tuberculosis NCTC 8337 (13) Mycobacterium tuberculosis H37Rv (14) Mycobacterium tuberculosis ATCC 25177 (15) Mycobacterium tuberculosis ATCC 358 1 8 (16) Mycobacterium tuberculosis ATCC 35828 Figure 3 shows the heterozygous reaction diagram of non-tuberculous mycobacteria target bacteria (1-1 9) and non-target bacteria (20-32) (1) Mycobacterium tuberculosis CCUG 20993 ® (2) Mycobacterium avium BCRC 15441 (3) Mycobacterium tuberculosis CCUG 35749 (4) Mycobacterium tuberculosis BCRC 1 5320 (5) Mycobacterium tuberculosis CCUG 29062 (6) Mycobacterium gordon CCUG 21801 (7) Mycobacterium tuberculosis CCUG 28005 (8) Kansas Mycobacterium CCUG 27785 (9) Mycobacterium lanatus CCUG 47901 I46866.doc -36· 201142038 (10) Mycobacterium marinum ATCC 25039 (11) Mycobacterium fortuitosis CCUG 28009 (12) Mycobacterium phlei CCUG 27976 (13) Mycobacterium tuberculosis CCUG 29045 (14) Mycobacterium tuberculosis CCUG 29114 (15) Mycobacterium smegmatis ATCC 19420 (16) Mycobacterium tuberculosis NCTC 10829 (17) Mycobacterium phlei CCUG 42429 • ( 18) Mycobacterium ulcerans ATCC 25899 (19) Mycobacterium phlei ATCC 19973 (20) Mycobacterium Μ CCUG 37673 (21) Mycobacterium Μ. α/vez· CCUG 3 7585 (22) Mycobacterium M CCUG 39146 (23) Mycobacterium M. asiaticum MB 03 1 (24) Mycobacterium M. e/nke CCUG 39504 (25) Mycobacterium M. c/z/or<9/?/ze«o/z_cwwBCRC 13726
(26) 分枝桿菌 M. coV/weWb CCUG 375 1 3 (27) 分枝桿菌 M.山16395 (28) 分枝桿菌 M. ga山-wm CCUG 375 1 5 (29) 分枝桿菌 M. pw/wb CCUG 37668 (30) 分枝桿菌 M. /fl//ax CCUG 375 84 (31) 分枝桿菌 Μ//αναα«·5 MB 017 (32) 分枝桿菌 M. MB 691 146866.doc •37- 201142038 序列表 <110> 國立成功大學 <120>鑑定分枝桿菌之方法 <130> 無 <160 39 <170> Patentln version 3.3 <210> 1 <211> 21 <212> DNA <213> 人工序列 <220>(26) Mycobacterium M. coV/weWb CCUG 375 1 3 (27) Mycobacterium M. Mountain 16395 (28) Mycobacterium M. gashan-wm CCUG 375 1 5 (29) Mycobacterium M. pw /wb CCUG 37668 (30) Mycobacterium M. /fl//ax CCUG 375 84 (31) Mycobacterium Μ//αναα«·5 MB 017 (32) Mycobacterium M. MB 691 146866.doc •37 - 201142038 Sequence Listing <110> National Cheng Kung University <120> Method for identifying mycobacteria <130> No <160 39 <170> Patentln version 3.3 <210> 1 <211> 21 <212> ; DNA <213> Manual Sequence <220>
<221> 變異 <222〉(1)..(21) <223> 丫為(:或t <400> 1 21 25 yctgtagtgg gcgagagccg g <210> 2 <211> 25 <212> DNA <213> 人工序列 <220〉 <221>變異 <222〉(1)..(25) <223> r 為 a或g <400〉 2 cacactgttg ggtcctgagr caaca <210> 3 <211> 18 <212> DNA <213> 人工序列 <400> 3 gaaaacgccc caactggt 18 <210〉 4 <211> 21 146866.doc 201142038 <212> DNA <213> 人工序列 <400> 4 atgacaacaa agttggccac c <210〉 5 <211> 20 <212〉DNA <213> 人工序列 <400> 5 ggtcaacggt gcggttctgg<221> Variation <222>(1)..(21) <223> 丫 is (: or t <400> 1 21 25 yctgtagtgg gcgagagccg g <210> 2 <211> 25 <212> ; DNA <213> Artificial sequence <220>221>Variation<222>(1)..(25)<223> r is a or g <400> 2 cacactgttg ggtcctgagr caaca <210> 3 <211> 18 <212> DNA <213> artificial sequence <400> 3 gaaaacgccc caactggt 18 <210> 4 <211> 21 146866.doc 201142038 <212> DNA <213><400> 4 atgacaacaa agttggccac c <210> 5 <211> 20 <212>DNA <213> Artificial sequence <400> 5 ggtcaacggt gcggttctgg
<210> 6 <211> 20 <212> DNA <213> 人工序列 <400〉 6 ggtcaacggt acggttctgg <210> 7 <211> 23 <212> DNA <213> 人工序列 <400〉 7 gaaggtctgt aacgaacagc tga <210〉 8 <211> 23 <212> DNA <213> 人工序列 <400〉 8 gaaggtctgt aatgaacagc tga <210> 9 <211> 22 <212〉DNA <213> 人工序列 <400〉 9 cgaaagtcgt tgtgaacaag gc 146866.doc 201142038 <210> 10 <211> 22 <212〉DN A <213> 人工序列 <400> 10 cgaaagttgt tgtgaacaag gc <210〉 11 <211> 18 <212> DNA <213> 人工序列 <400> 11 cctggccgct gtgatctc<210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 ggtcaacggt acggttctgg <210> 7 <211> 23 <212> DNA <213> Artificial sequence<213> 400> 7 gaaggtctgt aacgaacagc tga <210> 8 <211> 23 <212> DNA <213> Artificial sequence <400> 8 gaaggtctgt aatgaacagc tga <210> 9 <211> 22 <212> DNA <213> Artificial sequence <400> 9 cgaaagtcgt tgtgaacaag gc 146866.doc 201142038 <210> 10 <211> 22 <212>DN A <213> Artificial sequence <400> 10 cgaaagttgt tgtgaacaag gc <;210> 11 <211> 18 <212> DNA <213> Manual Sequence <400> 11 cctggccgct gtgatctc
<210〉 12 <211> 18 <212> DNA <213〉人工序列 <400> 12 cctggccgcg gtgatctc <210〉 13 <211> 22 <212> DNA <213> 人工序列 <400〉 13<210> 12 <211> 18 <212> DNA <213>Artificial sequence <400> 12 cctggccgcg gtgatctc <210> 13 <211> 22 <212> DNA <213> Artificial sequence <;400> 13
gggtacgagt ggtctcaggt tt <210〉 14 <211> 22 <212〉DNA <213> 人工序列 <400〉 14 gggtatgagt ggtctcaggt tt <210〉 15 <211> 18 <212> DNA <213> 人工序列 146866.doc 18201142038Gggtacgagt ggtctcaggt tt <210> 14 <211> 22 <212>DNA <213> Artificial sequence <400> 14 gggtatgagt ggtctcaggt tt <210> 15 <211> 18 <212> DNA <213>; Artificial sequence 146866.doc 18201142038
<400> 15 gactttcgag tcggtggg <210〉 16 <211〉 18 <212> DNA <213> 人工序列 <400> 16 gactttcgcg tcggtggg <210〉 17 <211> 17 <212> DNA <213> 人工序列 <400> 17 ccgtggatgg gtagtcg <210〉 18 <211> 20 <212> DNA <213> 人工序列 <400〉 18 gttcccgtct gtaatggacg 18 17 20<400> 15 gactttcgag tcggtggg <210> 16 <211> 18 <212> DNA <213> Artificial sequence <400> 16 gactttcgcg tcggtggg <210> 17 <211> 17 <212> DNA <213> Artificial sequence <400> 17 ccgtggatgg gtagtcg <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 gttcccgtct gtaatggacg 18 17 20
<210〉 19 <211> 19 <212〉 DNA <213> 人工序列 <400> 19 cccgtggata ggtagtcgg 19 <210> 20 <211> 18 <212> DNA <213> 人工序列 <400〉 20 attcgattcc cccgccgt <210> 21 <211> 25 146866.doc 18 201142038 <212> DNA <213> 人工序列 <400> 21 cagcaagcaa gccagacaca ctatt <210> 22 <211> 20 <212> DNA <213> 人工序列 <400〉 22 gccgtgaagg gtcatcgtct<210> 19 <211> 19 <212> DNA <213> Artificial sequence <400> 19 cccgtggata ggtagtcgg 19 <210> 20 <211> 18 <212> DNA <213> Artificial sequence <400> 20 attcgattcc cccgccgt <210> 21 <211> 25 146866.doc 18 201142038 <212> DNA <213> Artificial sequence <400> 21 cagcaagcaa gccagacaca ctatt <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <400> 22 gccgtgaagg gtcatcgtct
<210> 23 <211> 19 <212> DNA <213> 人工序列 <400〉 23 gggtgcacaa cagcgaatg <210 24 <211> 19 <212> DNA <213> 人工序列 <400〉 24 tggacgaaag cctggtgac <210〉 25 <211> 19 <212> DNA <213> 人工序列 <400> 25 ttggtggagt gtgagccgt <210〉 26 <211> 19 <212> DNA <213> 人工序列 <400> 26 tagtggatgg aagccgggt 146866.doc 201142038 <210> 27 <211> 21 <212> DNA <213> 人工序列 <400> 27 gtggttggga tacatttcgc c<210> 23 <211> 19 <212> DNA <213> Artificial sequence <400> 23 gggtgcacaa cagcgaatg <210 24 <211> 19 <212> DNA <213> Artificial sequence < 400> 24 tggacgaaag cctggtgac <210> 25 <211> 19 <212> DNA <213> Artificial sequence <400> 25 ttggtggagt gtgagccgt <210> 26 <211> 19 <212> DNA <213> Manual sequence <400> 26 tagtggatgg aagccgggt 146866.doc 201142038 <210> 27 <211> 21 <212> DNA <213> Artificial sequence <400> 27 gtggttggga tacatttcgc c
<210〉 28 <211〉 19 <212> DNA <213> 人工序列 <400〉 28 tggtgggtgt cggcgtgtt <210> 30 <211> 19 <212> DNA <213> 人工序列<210> 28 <211> 19 <212> DNA <213> Artificial sequence <400> 28 tggtgggtgt cggcgtgtt <210> 30 <211> 19 <212> DNA <213> Artificial sequence
<210〉 29 <211> 20 <212> DNA <213> 人工序列 <400〉 29 ggggttctcg cctgtagtgg <400> 30 ttggtggagt gtgagccgt <210〉 31 <211> 22 <212> DNA <213> 人工序列 <400> 31 tgaggagctg gatcgctgta gt <210〉 32 <211> 20 <212〉DNA <213> 人工序列 146866.doc 201142038 <400〉 32 cacgaaacgc atcccaactg <210> 33 <211> 18 <212> DNA <213> 人工序列 <400〉 33 ccgccagcag ccttgtgc <210> 34 <211> 19 <212〉DNA <213> 人工序列<210> 29 <211> 20 <212> DNA <213> Artificial sequence <400> 29 ggggttctcg cctgtagtgg <400> 30 ttggtggagt gtgagccgt <210> 31 <211> 22 <212> DNA <213> Artificial sequence <400> 31 tgaggagctg gatcgctgta gt <210> 32 <211> 20 <212>DNA <213> Artificial sequence 146866.doc 201142038 <400> 32 cacgaaacgc atcccaactg <210> 33 <211> 18 <212> DNA <213> artificial sequence <400> 33 ccgccagcag ccttgtgc <210> 34 <211> 19 <212>DNA <213>
<400〉 34 tagtggatgg aagccgggt <210〉 35 <211> 20 <212> DNA <213> 人工序列 <400〉 35 gtgtgagttc ggcgtgttgt <210〉 36 <211> 21 <212〉DNA <213〉人工序列 <400> 36 acctcctttc taaggagcac c <210> 37 <211> 21 <212> DNA <213> 人工序列 <400〉 37 gatgctcgca accactatcc a <210> 38 <211> 19 146866.doc 201142038 <212〉DN A <213> 人工序列 <400> 38 tggttaacgc gctatccac <210> 39 <211> 19 <212> DNA <213> 人工序列 <400> 39 accaactctc gtgccttac<400> 34 tagtggatgg aagccgggt <210> 35 <211> 20 <212> DNA <213> Artificial sequence <400> 35 gtgtgagttc ggcgtgttgt <210> 36 <211> 21 <212>DNA <213>Artifical sequence <400> 36 acctcctttc taaggagcac c <210> 37 <211> 21 <212> DNA <213> Artificial sequence <400> 37 gatgctcgca accactatcc a <210> 38 <211> 19 146866.doc 201142038 <212>DN A <213> Artificial sequence <400> 38 tggttaacgc gctatccac <210> 39 <211> 19 <212> DNA <213> Artificial sequence <400>; 39 accaactctc gtgccttac
146866.doc146866.doc
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CN113667728A (en) * | 2021-07-23 | 2021-11-19 | 杭州圣庭医疗科技有限公司 | Rapid identification method for mycobacterium based on nanopore sequencer |
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CN110257535A (en) * | 2019-06-27 | 2019-09-20 | 大理大学 | Mycobacterium tuberculosis (Mtb) MLVA genotyping kit |
CN113667728A (en) * | 2021-07-23 | 2021-11-19 | 杭州圣庭医疗科技有限公司 | Rapid identification method for mycobacterium based on nanopore sequencer |
CN113667728B (en) * | 2021-07-23 | 2024-02-13 | 杭州圣庭医疗科技有限公司 | Quick mycobacterium identification method based on nanopore sequencer |
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