CN103114148B - Primer, probe and kit for fluorescence quantitative PCR (polymerase chain reaction) detection and parting of salmonella - Google Patents
Primer, probe and kit for fluorescence quantitative PCR (polymerase chain reaction) detection and parting of salmonella Download PDFInfo
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- CN103114148B CN103114148B CN201310068510.8A CN201310068510A CN103114148B CN 103114148 B CN103114148 B CN 103114148B CN 201310068510 A CN201310068510 A CN 201310068510A CN 103114148 B CN103114148 B CN 103114148B
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Abstract
The invention discloses a primer, a probe and a kit for fluorescence quantitative PCR (polymerase chain reaction) detection and parting of salmonella. The primer sequence is as shown in SEQ ID NO.1 and 2; the probe is as shown in SEQ ID NO.3 and 4; the bacteria to be tested are subjected to PCR augmentation by the primer and the probe; a 210bp strip is augmented; a salmonella positive sample enhanced at 640nm of wavelength is detected by fluorescence; and then the salmonella can be parted according to the melting temperature. The primer is significant in technical advantages, and has high specificity and sensitivity; and the main pathogenic bacteria (intestinal salmonella subspecies) and other subspecies of Bongor salmonella and intestinal salmonella can be conveniently parted. Compared with the technology disclosed by the invention, the primer can be conveniently and widely used for diagnosing infection of human and animal salmonella and bacterial food poisoning.
Description
Technical field
The present invention relates to fluorescence quantitative PCR detection reagent and the detection method of salmonella molecular diagnosis.This invention is the salmonella (6 subspecies of two kinds of salmonella, enteron aisle salmonella) of all strains of amplification effectively.In addition, high resolving power melting curve technology is divided into 3 groups salmonella easily: i) important pathogenic bacterium enteron aisle subspecies (69.5 ℃ of melting temperature (Tm)s), ii) person of outstanding talent's subspecies (64.2 ℃) and pair Ya Lisangla subspecies (63.8 ℃), iii) Bang Geer salmonella (54.8 ℃), Ying Dijia subspecies (57 ℃), salam subspecies (55.5 ℃), Arizona subspecies (54.5 ℃).
Background technology
Salmonella (Salmonella spp.) can cause the salmonellosis of the multiple different clinical manifestations of humans and animals, is also one of poisoning Etiological of human foods, all very important to medical science, veterinary science and public hygienics.Salmonella is divided into enteron aisle salmonella (S.enterica) and Bang Geer salmonella (S.bongori).Enteron aisle salmonella is divided into again 6 subspecies: enteron aisle subspecies (S.enterica enterica is abbreviated as S.enterica), salam subspecies (S.salamae), Arizona subspecies (S.arizonae), two Arizona subspecies (S.diarizonae), person of outstanding talent subspecies (S.houtanae) and Ying Dijia subspecies (S.indica).The diagnosis of Salmonella infection mainly depends on microbial culture, serum agglutination test and molecular diagnostic techniques.But, do not have single diagnostic method can detect easily and distinguish the salmonella (6 subspecies of two kinds of salmonella, enteron aisle salmonella) of all strains.
Summary of the invention
The object of the present invention is to provide a kind of fast, high specificity, highly sensitive, the quantitative PCR detection technique that is applicable to all strain salmonella of detection of clinical use.Based on highly sensitive melting curve technology, distinguish easily the different subspecies of Bang Geer salmonella and enteron aisle salmonella simultaneously.
Principle of the present invention and core are special, efficient primer and the probes that scientifically designs amplification and detect salmonella.When the primer that guarantees design efficiently increases the probe specific detection salmonella of salmonella, design, guarantee that this primer and probe do not increase and detect other similar bacterium.In addition, the probe of design is different with the base sequence degree of agreement of salmonella; Therefore, high resolving power melting point curve technology can show the different melting temperature (Tm)s of salmonella, and this can be easily for gene type.From GenBank(www.ncbi.nlm.nih.gov) obtain the sequence of ttrRSBCA of the following salmonella representative strains of subspecies (plant and) and Related Bacteria, by the method for Clustal Multiple Alignment Algorithm, all sequences is alignd:
Bang Geer salmonella, gene order number, AY578066;
Enteron aisle salmonella salam subspecies, gene order number, AY578070;
Enteron aisle salmonella Arizona subspecies, gene order number, CP000880;
The two Arizona of enteron aisle salmonella subspecies, gene order number, AY578069.1;
An enteron aisle salmonella person of outstanding talent subspecies, gene order number, AY578068;
Enteron aisle salmonella Ying Dijia subspecies, gene order number, AY578065);
The representative strains of enteron aisle salmonella enteron aisle subspecies: S.Gallinarum, gene order number, AM933173; S.Enteritidis, gene order number, AM933172; S.Weltevreden, gene order number, FR775221; S.Paratyphi, gene order number, CP000857; S.Typhi LT2, gene order number, AF282268; S.Dublin, gene order number, CP001144.
Primer and the probe of Fig. 1, Fig. 2, Fig. 3, Fig. 4 display design are as follows:
Upstream primer: 5 '-GATGTTYCTTAGCGCYTTACAGGC-3 ' (SEQ ID NO.1)
Downstream primer: 5 '-CCGACMGCGTAATATTTGGCTGAC-3 ' (SEQ ID NO.2)
Probe-1:5 '-ATACGCTTTCCGGCACGGCAAT-6-FAM-3 ' (SEQ ID NO.3)
Probe-2:5 '-LCRED640-CGTCRGTGGATTWCCGTCGCCCT-Phosphate-3 ' (SEQ ID NO.4)
The invention also discloses fluorescence quantitative PCR detection and the parting kit of salmonella, comprise 5 times of quantitative PCR reaction solutions, 22 μ l, and 5 times of primers of 22 μ l and the mixed solution of probe; Described primer and probe mixed solution are that the upstream primer, 5 μ M downstream primers, probe-1 of 1 μ M of 5 μ M is, probe-2 of 1 μ M containing concentration.
The present invention further provides salmonella classifying method, the method comprises the following steps:
(1), with above-mentioned primer and probe PCR amplification sample to be tested, amplify the band of 210bp, and fluoroscopic examination strengthens at 640nm wavelength is salmonella positive sample;
(2) after PCR completes, step (1) salmonella positive sample amplified production is carried out to high resolving power melting curve analysis, according to melting temperature (Tm), salmonella is carried out to somatotype, somatotype standard is: i) important pathogenic bacterium enteron aisle subspecies (69.5 ℃ of melting temperature (Tm)s), ii) person of outstanding talent's subspecies (64.2 ℃ of melting temperature (Tm)s) and pair Ya Lisangla subspecies (63.8 ℃ of melting temperature (Tm)s), iii) Bang Geer salmonella (54.8 ℃ of melting temperature (Tm)s), Ying Dijia subspecies (57 ℃ of melting temperature (Tm)s), salam subspecies (55.5 ℃ of melting temperature (Tm)s), Arizona subspecies (54.5 ℃ of melting temperature (Tm)s).
Salmonella PCR is specific to be determined: round pcr specificity of the present invention is guaranteed from three aspects.I) by Intergrated DNA Technology company, synthesized the representative strains of following 6 salmonella, this sequence contains the amplification region of PCR: Bang Geer salmonella, enteron aisle salmonella salam subspecies, enteron aisle salmonella Arizona subspecies, the two Arizona of enteron aisle salmonella subspecies, an enteron aisle salmonella person of outstanding talent subspecies, enteron aisle salmonella Ying Dijia subspecies, the representative strains Salmonella typhi of enteron aisle salmonella enteron aisle subspecies.By the PCR system (upstream primer, downstream primer, probe-1, probe-2) of design, synthetic salmonella is carried out to pcr amplification amplification; Ii) the DNA of the positive strain of 15 strain salmonella is carried out to pcr amplification; Iii) the nucleic acid (Escherichia coli, Pasteurella multocida and Pseudomonas aeruginosa) of negative control and other similar gram-negative bacteria is carried out to pcr amplification; Iv) observe the variation of above amplification object fluorescence intensity (640nm) in PCR process, and pcr amplification product is in the size of the band of agarose gel electrophoresis.Fluorescence occurs or strengthens at 640nm wavelength, shows positive; The pcr amplification product of salmonella shows the band of the 210bp of expection at agarose gel electrophoresis; Iii) the PCR product of amplification is checked order, the result of order-checking and the sequence of GenBank compare.Result shows, the not of the same race and subspecies of the amplification salmonella that the salmonella PCR of design is special, and other similar bacteriums of not increasing.
Determining of the sensitivity of salmonella PCR: the sequence of being synthesized the ttrRSBCA of salmonella and Related Bacteria by Intergrated DNA Technology.According to the DNA quantitative technique of molecular weight and absolute weight and the PicoGreen of synthetics, calculate the absolute number of the gene copy of the contained ttrRSBCA of synthetics.Subsequently, synthetics is diluted, prepare the dilution reagent of every 10 μ l syntheticss containing the ttrRSBCA of 10000 copies, 1000 copies, 100 copies, 10 copies.Salmonella with above-mentioned PCR system amplification containing different concns ttrRSBCA, determines that the present invention detects the sensitivity of salmonella according to this.Result shows, the salmonella ttrRSBCA that this invention can amplified reaction system 10 copies.
Somatotype is differentiated: after PCR completes, the amplified production of the not of the same race and subspecies of salmonella is carried out to high resolving power melting curve analysis, observe the not melting temperature (Tm) of homophyletic salmonella.Due to the primer specific of design the salmonella of all strains of increasing, and the probe of design and not of the same race and subspecies salmonella have difference (Fig. 3, Fig. 4) in various degree, the salmonella of homophyletic can not show different melting temperature (Tm)s like this, and easily for gene type.High resolving power melting curve technology is divided into 3 groups (Fig. 5) salmonella easily: i) important pathogenic bacterium enteron aisle subspecies (69.5 ℃ of melting temperature (Tm)s), ii) person of outstanding talent's subspecies (64.2 ℃ of melting temperature (Tm)s) and pair Ya Lisangla subspecies (63.8 ℃ of melting temperature (Tm)s), iii) Bang Geer salmonella (54.8 ℃ of melting temperature (Tm)s), Ying Dijia subspecies (57 ℃ of melting temperature (Tm)s), salam subspecies (55.5 ℃ of melting temperature (Tm)s), Arizona subspecies (54.5 ℃ of melting temperature (Tm)s).
Compared with prior art, the invention has the advantages that quick, special, to detect delicately all strains salmonella, and easily salmonella is carried out to basic molecule sizing.I) the requirements of process 2-4 days of the cultivation of traditional salmonella and separated (increment cultivation, selection and screening and culturing), evaluation, biochemical test etc., the present invention only needs 2-4 hour (approximately 90 minutes, PCR approximately 60 minutes, high resolving power melting curve analysis 5 minutes, result are judged 10 minutes to comprise DNA extraction); Ii) traditional microbial culture detects salmonella method, need that live, fertile bacterium, and this method only needs the nucleic acid of salmonella, does not need the bacterium living; Can frozen sample for salmonella of the present invention, detect, the requirement of sampling is reduced, and be easy to the popularization of this technology; Iii) the present invention is quantitative technology, and traditional salmonella isolation technique is only given and positive or negative qualitative results; Iv) the PCR diagnostic method of existing salmonella mainly increase to as if enteron aisle salmonella enteron aisle subspecies, and present method can increase 2 kinds of salmonella and 6 subspecies of enteron aisle salmonella; More have clinical meaning, the present invention can be easily distinguishes other subspecies of the main pathogenic fungi (enteron aisle salmonella enteron aisle subspecies) and Bang Geer salmonella, enteron aisle salmonella.Technology of the present invention is compared, and can be widely used in easily infection and the bacterial food poisoning of diagnosis people and animals salmonella.
Accompanying drawing explanation
Fig. 1: the upstream primer of salmonella quantitative fluorescent PCR.
The sequence of upstream primer is: 5 '-GATGTTYCTTAGCGCYTTACAGGC-3 ', containing 2 degenerate base designs (Y), 5 subspecies (salam subspecies, the Arizona subspecies of can effectively increase Bang Geer salmonella, enteron aisle salmonella, two Arizona subspecies, person of outstanding talent's subspecies, Ying Dijia subspecies), representative strains (S.Gallinarum, the S.Enteritidis of enteron aisle salmonella enteron aisle subspecies, S.Weltevreden, S.Paratyphi, S.Typhi LT2, S.Dublin).
Fig. 2: the downstream primer of salmonella quantitative fluorescent PCR.
The sequence of downstream trip primer is: 5 '-CCGACMGCGTAATATTTGGCTGAC-3 ', and containing 1 degenerate base design (M).Primer becomes antonymy with illustrated sequence.The confirmation such as pcr amplification, gel electrophoresis, can effectively increase 5 subspecies (salam subspecies of Bang Geer salmonella, enteron aisle salmonella of this sequence, Arizona subspecies, two Arizona subspecies, person of outstanding talent's subspecies, Ying Dijia subspecies), the representative strains (S.Gallinarum of enteron aisle salmonella enteron aisle subspecies, S.Enteritidis, S.Weltevreden, S.Paratyphi, S.Typhi LT2, S.Dublin).
Fig. 3: the 6-FAM probe of salmonella quantitative fluorescent PCR.
Probe-1(6-FAM probe) sequence is 5 '-ATACGCTTTCCGGCACGGCAAT-6-FAM-3 ', and the salmonella ttrRSBCA gene of homophyletic does not have misfitting of 1-3 base.The confirmation such as pcr amplification, gel electrophoresis, misfitting of probe and the less degree of PCR product, can effectively detect PCR product, and show different melting temperature (Tm)s, for molecule, shapes.This probe and probe-2 can be detected 5 subspecies (salam subspecies of Bang Geer salmonella, enteron aisle salmonella effectively, Arizona subspecies, two Arizona subspecies, person of outstanding talent's subspecies, Ying Dijia subspecies), representative strains (S.Gallinarum, the S.Enteritidis of enteron aisle salmonella enteron aisle subspecies, S.Weltevreden, S.Paratyphi, S.Typhi LT2, S.Dublin).
Fig. 4: the LCRed-640 probe of salmonella quantitative fluorescent PCR.
Probe-2(LCred-640 probe) sequence is
5 '-LCRED640-CGTCRGTGGATTWCCGTCGCCCT-Phosphate-3 ', containing a degenerate base, and the salmonella ttrRSBCA gene of homophyletic does not have misfitting of 1-2 base.The confirmation such as pcr amplification, gel electrophoresis, misfitting of probe and the less degree of PCR product, can effectively detect PCR product, and show different melting temperature (Tm)s, for molecule, shapes.This probe and probe-1 can be detected 5 subspecies (salam subspecies of Bang Geer salmonella, enteron aisle salmonella effectively, Arizona subspecies, two Arizona subspecies, person of outstanding talent's subspecies, Ying Dijia subspecies), representative strains (S.Gallinarum, the S.Enteritidis of enteron aisle salmonella enteron aisle subspecies, S.Weltevreden, S.Paratyphi, S.Typhi LT2, S.Dublin).
Fig. 5: the high resolving power melting curve analysis of not of the same race and subspecies salmonella.
After PCR completes, the DNA cloning product of 6 subspecies of salmonella Bang Geer kind and enteron aisle salmonella is carried out to high resolving power melting curve analysis, observe not of the same race and melting temperature (Tm)s subspecies.Probe-1 of design and probe-2 and the not sequence of homophyletic salmonella have base sequence in various degree to misfit (Fig. 3, Fig. 4).Therefore, high resolving power melting curve technology is divided into 3 groups salmonella easily: i) important pathogenic bacterium enteron aisle subspecies (69.5 ℃ of melting temperature (Tm)s), ii) person of outstanding talent's subspecies (64.2 ℃ of melting temperature (Tm)s) and pair Ya Lisangla subspecies (63.8 ℃ of melting temperature (Tm)s), iii) Bang Geer salmonella (54.8 ℃ of melting temperature (Tm)s), Ying Dijia subspecies (57 ℃ of melting temperature (Tm)s), salam subspecies (55.5 ℃ of melting temperature (Tm)s), Arizona subspecies (54.5 ℃ of melting temperature (Tm)s).The sample of negative control does not show melting peak.
Embodiment
The nucleotide sequence of PCR detection method the primer of the present invention and probe is as follows:
Upstream primer 5 ' GATGTTYCTTAGCGCYTTACAGGC-3 '
Downstream primer 5 ' CCGACMGCGTAATATTTGGCTGAC-3 '
Probe-1:5 '-ATACGCTTTCCGGCACGGCAAT-6-FAM-3 '
Probe-2 5 ' LCRED640-CGTCRGTGGATTWCCGTCGCCCT-Phosphate-3 ' detection method process of the present invention is as follows:
(1) prepare the standard quantitative reagent (standard) that PCR uses.By the sequence of the synthetic salmonella ttrRSBCA of Intergrated DNA Technology, this sequence contains the amplification region of PCR.According to the DNA quantitative technique of molecular weight and absolute weight and the PicoGreen of synthetics, calculate the gene copy number of the contained ttrRSBCA of synthetics.Subsequently, synthetics is diluted, prepare the dilution reagent of every 10 μ l syntheticss containing the ttrRSBCA of 10000 copies, 1000 copies, 100 copies, 10 copies, the standard quantitative reagent of using as PCR.
(2) prepare the DNA profiling of sample to be checked.The testing sample such as animal excrement, chicken meat sample is stored in Eppendorf pipe (20 ° of C or long-term-80 ° of C of preservation), and for DNA purifying, the DNA of purifying is eluted in 100 μ l T
10e
0.1(containing 10mM Tris-HCl, 0.1mM EDTA, pH8.5) is as the amplification template of PCR.
(3) pcr amplification system.The sample DNA template that the amplification system of 20 μ l comprises 10 μ l or quantitative criterion reagent (standard), 1xPCR damping fluid, 1 μ M upstream primer, 1 μ M downstream primer, probe-1 of 0.2 μ M, the commercialization Taq enzyme of probe-2 of 0.2 μ M, 2 units, 200 μ M dNTP.
(4) pcr amplification loop parameter.Pcr amplification comprises that the rigorous circulation of the height of 18 lapses of temperature (high-stringency step-down) and 25 owe rigorous fluorescence and obtain circulation (relaxed-stringency fluorescence acquisition).The rigorous circulation of height of 18 lapses of temperature: 6x15sec
95 ° of C, 60sec
74 ° of C; 9x15sec
95 ° of C, 60sec
72 ° of C; 3x15sec
95 ° of C, 10sec
70 ° of C, 15sec
c.25 owe rigorous fluorescence for 72 ° and obtain circulation: 25x15sec
95 ° of C, 8sec
58 ° of C, 10sec
65 ° of C, and15sec
72 ° of C.
(5) high resolving power melting curve analysis.After pcr amplification finishes, implement immediately high resolving power melting curve analysis, temperature rises to 80 ° of C from 38 ° of C, with 0.2 ° of C per second, increases progressively.Data analysis is the ratio of analyzing the fluorescence intensity of 640nm:530 nm (F2/F1).The first derivative value (d (F2/F1)/dt) of F2/F1 is read as the melting temperature (Tm) of this DNA.
(6) judgement of PCR result and quantitative analysis.The preparation process of DNA profiling is equipped with feminine gender and the positive control of DNA extraction, and pcr amplification object subsequently comprises the DNA profiling of testing sample, the feminine gender of DNA extraction and positive control, (every 10 μ l standard are containing 10 for quantitative standard reagent
4, 10
3, 10
2, 10
1the salmonella ttrRSBCA of copy).Pcr amplification efficiency of the present invention is high, and amplification is containing 10 effectively
1the standard of the salmonella ttrRSBCA of copy.
Embodiment 1: the pcr amplification of salmonella type strain
By the blood agar enteron aisle salmonella enteron aisle subspecies antityphoid sera type that 15 strains preserve of recovering, cultivate.Bacterial colony is positioned in 1XPBS damping fluid, is placed in 100 ℃ of water water-baths 5 minutes, and centrifugal 10 minutes of 4 ℃ of 10000rpm/min, get 10 μ l as pcr template.By the technology of the present invention, carry out pcr amplification and high resolving power melting curve analysis (concrete ins and outs as mentioned above).Result shows, the amplification enteron aisle salmonella enteron aisle subspecies antityphoid sera type of PCR energy differential high efficient, and the melting temperature (Tm) of 69 ° of C of appearance.
Embodiment 2: healthy dogs ight soil carries the detection of salmonella
The fresh excreta of collecting is frozen in-80 ℃ of refrigerators immediately, and rear taking-up 200mg (uses for nucleic acid extraction
kit method), get 10 μ l as pcr template.By the technology of the present invention, carry out pcr amplification and high resolving power melting curve analysis (concrete ins and outs and parameter are as mentioned above).In 127 faecal samples to be measured, 10 positive (10/127,7.9%).Melting curve analysis shows and confirms through gene sequencing, wherein 7 examples are enteron aisle salmonella enteron aisle subspecies (69 ℃ of melting temperature (Tm)s, 2 examples are an enteron aisle salmonella person of outstanding talent subspecies (64.2 ℃ of melting temperature (Tm)s), and 1 example is enteron aisle salmonella Arizona subspecies (54.5 ℃ of melting temperature (Tm)s).
Claims (3)
1. the fluorescence quantitative PCR detection of salmonella and the primer of somatotype and a probe, is characterized in that detecting primer and be:
Upstream primer: 5 '-GATGTTYCTTAGCGCYTTACAGGC-3 '
Downstream primer: 5 '-CCGACMGCGTAATATTTGGCTGAC-3 '
Described probe is:
Probe-1:5 '-ATACGCTTTCCGGCACGGCAAT-6-FAM-3 '
Probe-2:5 '-LCRED640-CGTCRGTGGATTWCCGTCGCCCT-Phosphate-3 '.
2. the fluorescence quantitative PCR detection of salmonella and a parting kit, comprise 5 times of quantitative PCR reaction solutions, 22 μ l, and 5 times of primers of 22 μ l and the mixed solution of probe; Described primer and probe mixed solution are that the upstream primer, 5 μ M downstream primers, probe-1 of 1 μ M of 5 μ M is, probe-2 of 1 μ M containing concentration.
3. a salmonella classifying method, it is for non-diagnostic purpose, and the method comprises the following steps:
(1), with claim 1 primer and probe PCR amplification sample to be tested, amplify the band of 210bp, and fluoroscopic examination strengthens at 640nm wavelength is salmonella positive sample;
(2) after PCR completes, step (1) salmonella positive sample amplified production is carried out to high resolving power melting curve analysis, according to melting temperature (Tm), salmonella is carried out to somatotype, somatotype standard is: i) important pathogenic bacterium enteron aisle subspecies melting temperature (Tm) is 69.5 ℃, ii) person of outstanding talent's subspecies, 64.2 ℃ of melting temperature (Tm)s; 63.8 ℃ of two Ya Lisangla subspecies melting temperature (Tm)s, iii) 54.8 ℃ of Bang Geer salmonella melting temperature (Tm)s, 57 ℃ of Ying Dijia subspecies melting temperature (Tm)s, 55.5 ℃ of salam subspecies melting temperature (Tm)s, 54.5 ℃ of Arizona subspecies melting temperature (Tm)s.
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| CN102443629A (en) * | 2011-11-02 | 2012-05-09 | 广州华银医学检验中心有限公司 | Fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella and detection method thereof |
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| CN102443629A (en) * | 2011-11-02 | 2012-05-09 | 广州华银医学检验中心有限公司 | Fluorescence quantitative PCR (polymerase chain reaction) detection kit for salmonella and detection method thereof |
Non-Patent Citations (4)
| Title |
|---|
| 杨柳.荧光定量RT_PCR检测沙门氏菌方法的建立.《中国实验诊断学》.2011,第15卷(第1期),17-20. |
| 王荣香.荧光定量PCR快速检测粪便中沙门氏菌方法的建立及应用.《放射免疫学杂志》.2012,第23卷(第3期),356-357. |
| 荧光定量PCR快速检测粪便中沙门氏菌方法的建立及应用;王荣香;《放射免疫学杂志》;20121231;第23卷(第3期);356-357 * |
| 荧光定量RT_PCR检测沙门氏菌方法的建立;杨柳;《中国实验诊断学》;20110131;第15卷(第1期);17-20 * |
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