CN101812518A - Multiple PCR detection kit for virulence factors of streptococcus suis and detection method thereof - Google Patents
Multiple PCR detection kit for virulence factors of streptococcus suis and detection method thereof Download PDFInfo
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Abstract
The invention discloses a multiple PCR detection kit for virulence factors of streptococcus suis and a detection method thereof, and belongs to the technical field of biology. The multiple PCR detection kit comprises nucleic acid shown by base sequences, such as SEQ ID NO:1 to SEQ ID NO:20. The detection method for the multiple PCR detection kit comprises the following steps of: 1, providing DNA of samples to be detected; 2, taking the kit, amplifying the DNA of the samples to be detected by adopting the conventional PCR method, detecting an amplified result by adopting an agarose gel electrophoresis method, and judging according to the result; and by using positive DNA of streptococcus suis 2 type as contrast, if the contrast proves that not all the strips are amplified, re-detecting, and if the contrast proves that all the strips are amplified, judging the result. The multiple PCR detection method established by the invention is specific and sensitive, and has the advantages of simpleness, convenience and quickness. The multiple PCR detection kit has reliable stability, and can be used for rapid detection of clinical samples and survey research on molecular epidemiology for the streptococcus suis.
Description
Technical field
The present invention relates to a kind of multiple PCR detection kit and detection method thereof of biological technical field, specifically is a kind of multiple PCR detection kit and detection method thereof of virulence factors of streptococcus suis.
Background technology
(Streptococcus suis is a kind of important infecting both domestic animals and human disease pathogen SS) to swine streptococcus, extensively distributes in the whole world, causes the meningitis of piglet, sacroiliitis, endocarditis, septicemia, the sudden death of pneumonia and weanling pig, but and infected person.According to the antigenic difference of thalline capsular polysaccharide, be divided into 35 serotypes (1～34 type and 1/2 type).SS2, SS7, SS9 are wherein most important pathogenic strainss, and be especially popular the widest with SS2, pathogenic the strongest.1991 in Guangdong Province first isolation identification break out the people in Jiangsu and some areas, Sichuan respectively in 1998 and 2005 subsequently and infect SS2 and causing death to SS2.
Swine streptococcus is divided into pathogenic strain and non-pathogenic strain.Pathogenic strain causes serious clinical symptom such as pig meningitis, sacroiliitis, endocarditis, septicemia, pneumonia, can cause the pig sudden death, and can cause people's infection and cause death; Non-pathogenic strain is common in the respiratory tract of normal pig, does not cause clinical symptom.Studies show that pathogenic its virulence factor that depends on of swine streptococcus, the main virulence factor of swine streptococcus comprises: capsular polysaccharide (cps), muramidase-released protein (mrp), extracellular factor (epf), glutamate dehydrogenase (gdh), fibronectin/fibrinogen conjugated protein (fbps), hemolysin (sly), virulence correlated series (orf2), glutamate dehydrogenase (gdh), glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene.Wherein, the cps gene can be used for distinguishing the serotype of swine streptococcus, and the gdh gene is owing to there are the characteristics of very low mutations in epithelial and high conservative often to be used as the diagnostic antigen of streptococcus suis infection, and gdh and cps are again virulence factors simultaneously.The pathogenic power of swine streptococcus depends on that its virulence factor, particularly mrp and epf Chang Zuowei judge one of pathogenic index.America and Europe etc. country finds the several genes type to the existing report of the distribution situation of part virulence factor, and the strongest pathogenic genotype is cps2/epf+/mrp+/sly+, and the research of domestic this respect is also few.
Find that through literature search publication number is that the Chinese invention patent application of CN1896279A has disclosed a kind of fast inspection reagent kit for streptococcus suis 2-type triple PCR, but this test kit can not detect the somatotype of swine streptococcus 7 types and 9 types to prior art.In recent years, though there is not the caused Streptococcus suis outbreak of epidemic of SS7, SS9, but the report that is separated to SS7 or SS9 in the sick pig body is arranged all both at home and abroad, it is pathogenic to studies show that further SS7, SS9 have, during polyinfection SS2 cause a disease or outbreak of epidemic in the performance synergy, therefore can to contain the multiple PCR detection kit of 7 kinds of main virulence factors of swine streptococcus (mrp, epf, sly, gdh, gapdh, orf2 and fbps) and 3 kinds of main pathogenic serotypes of swine streptococcus (2,7,9 type) very necessary in development.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of multiple PCR detection kit and detection method thereof of virulence factors of streptococcus suis is provided.The multi-PCR detection method that the present invention set up is special, sensitive, and has simple and efficient advantage; Test kit stability is reliable, can be used for the rapid detection of clinical sample and the Molecule Epidemiology Investigation research of swine streptococcus.
First aspect the present invention relates to a kind of multiple PCR detection kit of virulence factors of streptococcus suis, and this test kit comprises following component: 2 * PCR core reagent pre-composition, streptococcus suis 2,7,9 type positive control dnas, DL2000 molecular weight be with reference to solution, ddH
2O and following 10 kinds of primers are right:
Primer is to 1:
The upstream primer of base sequence shown in SEQ ID NO:1,
The downstream primer of base sequence shown in SEQ ID NO:2;
Primer is to 2:
The upstream primer of base sequence shown in SEQ ID NO:3,
The downstream primer of base sequence shown in SEQ ID NO:4;
Primer is to 3:
The upstream primer of base sequence shown in SEQ ID NO:5,
The downstream primer of base sequence shown in SEQ ID NO:6;
Primer is to 4:
The upstream primer of base sequence shown in SEQ ID NO:7,
The downstream primer of base sequence shown in SEQ ID NO:8;
Primer is to 5:
The upstream primer of base sequence shown in SEQ ID NO:9,
The downstream primer of base sequence shown in SEQ ID NO:10;
Primer is to 6:
The upstream primer of base sequence shown in SEQ ID NO:11,
The downstream primer of base sequence shown in SEQ ID NO:12;
Primer is to 7:
The upstream primer of base sequence shown in SEQ ID NO:13,
The downstream primer of base sequence shown in SEQ ID NO:14;
Primer is to 8:
The upstream primer of base sequence shown in SEQ ID NO:15,
The downstream primer of base sequence shown in SEQ ID NO:16;
Primer is to 9:
The upstream primer of base sequence shown in SEQ ID NO:17,
The downstream primer of base sequence shown in SEQ ID NO:18;
Primer is to 10:
The upstream primer of base sequence shown in SEQ ID NO:19,
The downstream primer of base sequence shown in SEQ ID NO:20.
Second aspect the invention still further relates to a kind of detection method of aforementioned agents box, comprises the steps:
Described judge according to the result be specially, with the positive DNA of streptococcus suis 2-type in contrast,, then detect again if contrast does not amplify following whole band; If contrast amplifies following whole band, then the result judges as follows:
If amplify 387bp and 688bp, then the gdh and the cps2J positive are streptococcus suis 2-type,
If amplify 251bp and 688bp, then the gdh and the cps7H positive are swine streptococcus 7 types,
If amplify 507bp and 688bp, then the gdh and the cps9D positive are swine streptococcus 9 types,
If amplify 316bp, the mrp positive then,
If amplify 626bp, the epf positive then,
If amplify 720bp, the fbps positive then,
If amplify 571bp, the gapdh positive then,
If amplify 858bp, the orf2 positive then,
If amplify 443bp, then the sly positive;
Do not have corresponding band, then be judged as feminine gender.
Preferably, the amplification parameter is specially in the described PCR method: 95 ℃ of 5min, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 36 circulations, 72 ℃ of 8min.
Compared with prior art, the present invention has following beneficial effect: the multi-PCR detection method that the present invention set up, when finishing 2,7,9 somatotypes, can realize detection to its 7 main virulence factors, by with the high specificity that relatively proves PCR reaction of other encountered pathogenic bacterias; The susceptibility experimental result shows that the PCR of single target gene detects lower limit and can reach 2.69 * 10
2CFU is limited to 2.69 * 10 under three multiplex PCR systems are detectable
3CFU proves that this method is highly sensitive, has reduced the probability of false negative and omission.The multi-PCR detection method that the present invention set up is not only special, sensitive, 3 individual system and many to combination of primers after, have simple and efficient advantage; And test kit stability is reliable, can be used for the rapid detection of clinical sample and the Molecule Epidemiology Investigation research of swine streptococcus.
Description of drawings
Fig. 1 declares electrophorogram for test kit detected result electrophoresis;
Fig. 2 is all virulence factor substance pcr amplification results of streptococcus suis 2-type positive control;
Fig. 3 is a different bacterium amount pcr amplification product electrophorogram; Wherein A and B are respectively different bacterium amount cps2J gene substance and PCR III multiplex PCR amplified production electrophorogram;
Fig. 4 is a different bacterium pcr amplification product electrophorogram, and wherein A and B are respectively different bacterium cps2J gene substance and PCR III multiplex PCR amplified production electrophorogram;
Fig. 5 is the dna molecular amount standard of DL2000Marker.
Specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Test kit and detection method that present embodiment relates to are as follows:
1, test kit
The multiple PCR detection kit of present embodiment comprises 10 frozen pipes, 50 PCR reaction tubess and specification sheets.10 frozen pipes are specially: code name is respectively the frozen pipe of M (2), C2, C7, C9, D, H, P1, P2, P3, and frozen pipe M is equipped with 2 * PCR core reagent pre-composition (dNTP, Taq archaeal dna polymerase mixture and MgCl
2Solution, commercial), frozen pipe C2, C7 and C9 are equipped with 30 μ l streptococcus suis 2s, 7,9 type positive control dna solution respectively, and frozen pipe D is equipped with the DL2000 molecular weight of 0.5ml with reference to solution (commercial), and frozen pipe H is equipped with the ddH of 1.5ml
2O, frozen pipe P1, P2, P3 are equipped with the powder mixture of PCR system I, II and III the primer respectively; All frozen Guan Jun are stored in-20 ℃.
2, the detection method of test kit
Primer among the P1 is:
The primer of amplification gene cps2J is to being C2U-I (SEQ ID NO:1), C2D-I (SEQ ID NO:2),
The primer of amplification gene cps7H is to being C7U-I (SEQ ID NO:3), C7D-I (SEQ ID NO:4),
The primer of amplification gene cps9D is to being C9U-I (SEQ ID NO:5), C9D-I (SEQ ID NO:6),
The primer of amplification gene gdh is to being g1-I (SEQ ID NO:7), g2-I (SEQ ID NO:8);
Primer among the P2 is:
The primer of amplification gene mrp is to being m1-II (SEQ ID NO:9), m2-II (SEQ ID NO:10),
The primer of amplification gene epf is to being e1-II (SEQ ID NO:11), e2-II (SEQ ID NO:12),
The primer of amplification gene fbps is to being f1-II (SEQ ID NO:13), f2-II (SEQ ID NO:14);
Primer among the P3 is:
The primer of amplification gene gapdh is to being h1-III (SEQ ID NO:15), h2-III (SEQ ID NO:16),
The primer of amplification gene orf2 is to being o1-III (SEQ ID NO:17), o2-III (SEQ ID NO:18),
The primer of amplification gene sly is to being s1-III (SEQ ID NO:19), s2-III (SEQ ID NO:20).
The positive control operation is identical, when streptococcus suis 2,7, the detection of 9 type somatotypes, and solution among template difference corresponding frozen pipe C2, C7 and the C9; The pipe of each somatotype adds 2,7,9 types contrast DNA respectively, and the purpose band that amplification is at last come out is to two kind in gdh and 2,7,9 types.
(1) the streptococcus suis 2-type positive control dna can amplify cps2J, gdh, mrp, epf, fbps, gapdh, the whole fragments of orf2, sly, positive control as the virulence factor judgement, if positive control can amplify whole bands, 3 multiplex PCR system results judge as follows:
PCR?I
If amplify 387bp and 688bp, then judge the gdh and the cps2J positive, be streptococcus suis 2-type (Figure 1A, No. 1 swimming lane);
If amplify 251bp and 688bp, then judge the gdh and the cps7H positive, be swine streptococcus 7 types (Figure 1A, No. 2 swimming lanes);
If amplify 507bp and 688bp, then judge the gdh and the cps9D positive, be swine streptococcus 9 types (Figure 1A, No. 3 swimming lanes);
Shown in Figure 1A,
No. 12 bands appear in swimming lane, are cps2J (387bp)+gdh (688bp);
No. 22 bands appear in swimming lane, are cps7H (251bp)+gdh (688bp);
No. 32 bands appear in swimming lane, are cps9D (507bp)+gdh (688bp);
PCRII
If amplify 316bp, then judge the mrp positive;
If amplify 626bp, then judge the epf positive;
If amplify 720bp, then judge the fbps positive;
Shown in Figure 1B, No. 13 bands appear in swimming lane, are mrp (316bp)+epf (626bp)+fbps (720bp);
PCRIII
If amplify 571bp, then judge the gapdh positive;
If amplify 858bp, then judge the orf2 positive;
If amplify 443bp, then judge the sly positive;
Shown in Fig. 1 C, No. 13 bands appear in swimming lane, are gapdh (571bp)+orf2 (858bp)+sly (443bp);
Do not have corresponding band or stripe size and misfit, then be judged as feminine gender;
(2) the streptococcus suis 2-type positive control dna can amplify cps2J, gdh, mrp, epf, fbps, gapdh, the whole fragments of orf2, sly, positive control as the virulence factor judgement, do not amplify at positive control under the situation of whole bands, need detect again and judge, as shown in Figure 2, among Fig. 2, each swimming lane is 1-cps2J, 2-mrp, 3-epf, 4-fbps, 5-gdh, 6-orf2,7-sly, 8-gapdh.
The material that present embodiment relates to is as follows:
Streptococcus aureus (ATCC 29213), intestinal bacteria (ATCC25922), streptococcus agalactiae (ATCC10556), enterococcus faecalis (ATCC29212) and campylobacter jejuni (ATCC33560) can be available from commercial companies, cover gene biological Science and Technology Ltd. again as Shanghai, CompanyAddress: in No. 229 Fudan University in Handan, Shanghai City road.
Sichuan strain isolated ZY05719 (streptococcus suis 2-type), Jiangsu strain isolated HA9801 (streptococcus suis 2-type) is open in many pieces of articles, as " Shen Yan, Hua Xiuguo, Cui Li etc. the representative strain isolateds of streptococcus suis 2-type two infect behind the miniature pigs influence [J] to the partial immunity index. journal of animal science and veterinary medicine, 2008,39 (12); 1721～1730 " open in, relevant bacterial strain can be obtained by disclosed commercial channel, as Qingdao China's animal health and epidemiology center, address: No. 369 postcodes in Nanjing Road, Qingdao City: 266032.
Streptococcus suis 2-type reference strain (S735), swine streptococcus 7 type reference strain (8704), swine streptococcus 9 type reference strain (2083) are available from Qingdao China's animal health and epidemiology center.Wherein streptococcus suis 2-type reference strain (S735) discloses (Marie-Claude Jobin in article, Julie Brassard, .Acquisition of Host Plasmin Activity by the Swine Pathogen Streptococcus suisSerotype 2 such as Sylvain Quessy, Infection and Immunity, January 2004, p.606～610, Vol.72, No.1); The SH29 strain is open in " Zhao Ran, Sun Jianhe, Lu Chengping, multiplex PCR evaluation streptococcus suis 2-type virulent strain, Shanghai Communications University's journal: agricultural sciences version, 2006 24 6 phases of volume, 503～506 ".
Swine streptococcus is inoculated in the 2mlTHB broth culture, and 37 ℃ of shaking culture are spent the night; Other microbionations are in 2ml meat soup enrichment medium, and 37 ℃ of shaking culture are spent the night.Carry out following experiment afterwards:
1, sensitivity test
SH29 strain inoculation 2ml THB broth culture, 37 ℃ of shaking culture are spent the night, and get after the centrifugal back of 1.5ml bacterium liquid washes 2 times with the PBS of 0.01mol/mL, make 10 times of continuous serieses and dilute, after carrying out plate count, get each dilution bacterium liquid and carry out the sensitivity test of substance and multiplex PCR.After the dilution streptococcus suis 2-type SH29 of difference strain carried out plate count, the concentration of extrapolating former SS2 bacterium liquid was 2.69 * 10
7CFU/mL.Extract DNA and carry out the sensitivity test of substance and multiplex PCR, the result shows that substance PCR susceptibility can reach 10
2CFU, multiplex PCR susceptibility can reach 10
3CFU (Fig. 3 A, 3B),
Among Fig. 3 A, swimming lane 1 to 8 pairing concentration is respectively: 2.69 * 10
7CFU, 2.69 * 10
6CFU, 2.69 * 10
5CFU, 2.69 * 10
4CFU, 2.69 * 10
3CFU, 2.69 * 10
2CFU, 2.69 * 10
1CFU, 2.69 * 10
0CFU;
Among Fig. 3 B, swimming lane 1 to 6 pairing concentration is respectively: 2.69 * 10
5CFU, 2.69 * 10
4CFU, 2.69 * 10
3CFU, 2.69 * 10
2CFU, 2.69 * 10
1CFU, 2.69 * 10
0CFU.
2, specificity test
With the SS2 positive control is template, the DNA that uses streptococcus aureus (available from ATCC), intestinal bacteria, campylobacter jejuni, enterococcus faecalis, streptococcus agalactiae respectively is a template, carries out the substance PCR of cps2J gene respectively and adopts the PCR method of setting up to increase.Except that positive template can amplify corresponding band, with streptococcus aureus (ATCC), intestinal bacteria, campylobacter jejuni, enterococcus faecalis, streptococcus agalactiae DNA is template detection, and the result shows that the PCR of single target gene and multiplex PCR all do not go out to reveal specific amplified band (Fig. 4 A, 4B).
Fig. 4 A is a different bacterium cps2J substance PCR electrophorogram as a result; Wherein each swimming lane is:
M.DL2000,1.2 type positive controls, 2.HA9801,3.8704 (SS7), 4. streptococcus aureus (ATCC), 5. bacillus coli DH 5 alpha, 6. campylobacter jejuni;
Fig. 4 B is a different bacterium PCRII multiplex PCR electrophorogram as a result; Wherein each swimming lane is:
M.DL2000,1.2 type positive controls, 2.HA9801,3.ZY05719,4. streptococcus aureus (ATCC), 5. bacillus coli DH 5 alpha, 6. campylobacter jejuni, 7. enterococcus faecalis, 8. streptococcus agalactiae.
Claims (3)
1. the multiple PCR detection kit of a virulence factors of streptococcus suis is characterized in that, comprises following component: 2 * PCR core reagent pre-composition, streptococcus suis 2,7,9 type positive control dnas, DL2000 molecular weight be with reference to solution, ddH
2O and following 10 kinds of primers are right:
Primer is to 1:
The upstream primer of base sequence shown in SEQ ID NO:1,
The downstream primer of base sequence shown in SEQ ID NO:2;
Primer is to 2:
The upstream primer of base sequence shown in SEQ ID NO:3,
The downstream primer of base sequence shown in SEQ ID NO:4;
Primer is to 3:
The upstream primer of base sequence shown in SEQ ID NO:5,
The downstream primer of base sequence shown in SEQ ID NO:6;
Primer is to 4:
The upstream primer of base sequence shown in SEQ ID NO:7,
The downstream primer of base sequence shown in SEQ ID NO:8;
Primer is to 5:
The upstream primer of base sequence shown in SEQ ID NO:9,
The downstream primer of base sequence shown in SEQ ID NO:10;
Primer is to 6:
The upstream primer of base sequence shown in SEQ ID NO:11,
The downstream primer of base sequence shown in SEQ ID NO:12;
Primer is to 7:
The upstream primer of base sequence shown in SEQ ID NO:13,
The downstream primer of base sequence shown in SEQ ID NO:14;
Primer is to 8:
The upstream primer of base sequence shown in SEQ ID NO:15,
The downstream primer of base sequence shown in SEQ ID NO:16;
Primer is to 9:
The upstream primer of base sequence shown in SEQ ID NO:17,
The downstream primer of base sequence shown in SEQ ID NO:18;
Primer is to 10:
The upstream primer of base sequence shown in SEQ ID NO:19,
The downstream primer of base sequence shown in SEQ ID NO:20.
2. the detection method of the multiple PCR detection kit of a virulence factors of streptococcus suis according to claim 1 is characterized in that, comprises the steps:
Step 1 provides the DNA of testing sample;
Step 2 is got test kit, adopts the DNA of conventional PCR method amplification testing sample, adopts agarose gel electrophoresis method to detect amplification, judges according to the result;
Described judge according to the result be specially, with the positive DNA of streptococcus suis 2-type in contrast,, then detect again if contrast does not amplify following whole band; If contrast amplifies following whole band, then judge as follows:
If amplify 387bp and 688bp, then the gdh and the cps2J positive are streptococcus suis 2-type,
If amplify 251bp and 688bp, then the gdh and the cps7H positive are swine streptococcus 7 types,
If amplify 507bp and 688bp, then the gdh and the cps9D positive are swine streptococcus 9 types,
If amplify 316bp, the mrp positive then,
If amplify 626bp, the epf positive then,
If amplify 720bp, the fbps positive then,
If amplify 571bp, the gapdh positive then,
If amplify 858bp, the orf2 positive then,
If amplify 443bp, then the sly positive;
Do not have corresponding band, then be judged as feminine gender.
3. the detection method of the multiple PCR detection kit of virulence factors of streptococcus suis according to claim 2 is characterized in that, the amplification parameter is specially in the described PCR method: 95 ℃ of 5min, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 36 circulations, 72 ℃ of 8min.
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| CN109666750A (en) * | 2018-12-19 | 2019-04-23 | 山东省农业科学院畜牧兽医研究所 | A kind of multiple real time fluorescence PCR detection primer composition and detection method identifying Streptococcus suis and pig pasteurella multocida |
| CN111534611A (en) * | 2019-11-12 | 2020-08-14 | 广州微芯生物科技有限公司 | Fluorescent quantitative PCR method for detecting toxigenic streptococcus suis and corresponding kit |
| CN112941211A (en) * | 2021-03-17 | 2021-06-11 | 哈尔滨瀚邦医疗科技有限公司 | Multiplex fluorescence quantitative PCR detection kit for streptococcus suis type 2 virulence genes and detection method thereof |
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