CN103911430A - Fluorescence labeling multiplex PCR detection kit for Clostridium difficile - Google Patents
Fluorescence labeling multiplex PCR detection kit for Clostridium difficile Download PDFInfo
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- CN103911430A CN103911430A CN201410059155.2A CN201410059155A CN103911430A CN 103911430 A CN103911430 A CN 103911430A CN 201410059155 A CN201410059155 A CN 201410059155A CN 103911430 A CN103911430 A CN 103911430A
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- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention provides a fluorescence labeling multiplex PCR detection kit for identification of a Clostridium difficile strain and analysis of toxin genes. The kit comprises 6 pairs of fluorescently-labeled primers used for detecting the Clostridium difficile strain. The 6 pairs of fluorescently-labeled primers comprise specific primers of specific triosephosphate-isomerase (tpi) gene, toxin A gene (tcd A), toxin B gene (tcd B), tcd C gene (tcd C), binary toxin A gene (cdt A) and binary toxin B gene (cdt B) of the Clostridium difficile strain. Amplification results are detected through capillary electrophoresis. Thus, existence of Clostridium difficile can be rapidly identified and toxin typing can be rapidly determined.
Description
Technical field
The present invention relates to comprise, for detection of the test kit of clostridium difficile (Clostridium difficile) bacterial strain or poisonous clostridium difficile strain, and detect the method for the clostridium difficile of qualification in the sample toxin somatotype that has that it's too late.
Background technology
Clostridium difficile (Clostridium difficile) is gram-positive product gemma anaerobic bacillus(cillus anaerobicus).Itself does not have aggressive clostridium difficile, part is produced malicious bacterium and is caused even lethality pseudomembranous enterocolitis of antibiotic-associated diarrhea, colitis by the A that excretes poison, toxin B and binary toxin, be referred to as C. difficile infection (Clostridium difficile infection, CDI).Hospital acquired infection is suffered from diarrhoea in clear and definite cause of disease, the most common with clostridium difficile; In the antibiotic-associated diarrhea cause of disease, clostridium difficile also accounts for 20%～30%; Pseudomembranous enterocolitis is almost 100% by due to clostridium difficile.In the U.S., C. difficile infection is first reason that causes Nosocomial Diarrhea.
Although this microorganism is used as for a long time to a kind of pathogenic bacteria of the not common infection that can lead to grave consequences always, recent years, there is variation in the feature of C. difficile infection., over 5 years, there is the report of the increase such as severity and lethality rate of a lot of disease incidents that cause about clostridium difficile, disease in past.This EPDML change is relevant with the clostridium difficile strain that has occurred a kind of new high virulence, this bacterial strain is called as clostridium difficile NAP1(North American pulsed-field type1), PCR ribotype 027(polymerase chain reaction ribotype027) popular bacterial strain, more unexistent features of common clostridium difficile before this bacterial strain has, as the generation of toxin T cdA and TcdB increases, carry and produce the third toxin, be again the gene of binary toxin (binary toxin), and occur that in tcd C gene, 18 base pairs lack.The resistance of quinolone antibiotic is increased.Due to the output of NAPI/027 bacterial strain toxin A, B, more common bacterial strain is high 16 times and 23 times respectively, therefore causes that serious clostridium difficile is diseases related.
Clostridium difficile detect have direct ight soil toxin determination comprise cytotoxin measure (CTA), enzyme immunoassay and.In CTA, the detection of toxin B is considered to the gold standard that clostridium difficile detects.But this test needs 48～72h, and requirement for experiment condition is higher, and the reliability of its result changes also very large between different laboratories.
The method that fluorescent quantitation detects detects becomes the main flow detection technique that clostridium difficile detects gradually because it is quick, sensitive.Rough estimates, (the Food and Drug Administration of food and drug administration, FDA) the detection by quantitative product of certification has the Gene-Ohm detection system of BD company, the proGastro detection system of Gen-Probe company, the Xpert detection system of Cepheid company, the Portrait detection system of Great Basin company, the Simplexa detection system of Focus DX company.These detection systems are removed Xpert and are all only detected Clostridium difficile toxin B (tcd B) gene.And the Xpert detection system of Cepheid company detects toxin B(tcdB simultaneously), tcdC genetically deficient (tcd C deletion), binary toxin (Binary Toxin).
The scheme that Chinese patent application 201310108285.6 provides sensitivity in the time detecting single-gene can be accomplished 1 copy, but does not provide technology enlightenment how to carry out multiplex PCR amplification.
The detection system that Chinese patent application 201310316848.0 provides can detect four locus simultaneously, Clostridium difficile toxin A (tcdA), toxin B(tcdB), binary toxin A(cdtA) and binary toxin B(cdtB), sensitivity is minimum is 3pg/ μ l.It is low that the visible multiple quantitative PCR remolding sensitivity substance expanding detects.
Summary of the invention
The object of the invention is: provide one can detect the specific triosephosphate isomerase of clostridium difficile bacterial classification (triosephosphate isomerase simultaneously, tpi) gene, toxin A gene (tcdA), toxin B(tcd B), tcdC gene (tcdC), binary toxin A(cdtA) and binary toxin B(cdtB) composite amplification reagent kit, need it to have advantages of that somatotype is accurate, detectability is low.
Technical scheme:
A kind of fluorescent mark multiplex PCR clostridium difficile detection kit, include triosephosphate isomerase (triosephosphate-isomerase, tpi) gene, toxin A gene (tcdA), toxin B gene (tcd B), tcdC gene (tcdC), binary toxin A gene (cdtA) and the corresponding primer of binary toxin B gene (cdtB) for the clostridium difficile that increases.
Triosephosphate isomerase is clostridium difficile bacterial classification specific proteins, identifies in sample whether contain clostridium difficile by detecting having or not of triosephosphate isomerase (triosephosphate-isomerase, tpi) gene.Design Auele Specific Primer increase respectively toxin A gene (tcdA), toxin B gene (tcd B), tcdC gene (tcdC), binary toxin A gene (cdtA) and binary toxin B gene (cdtB), whether whether purely in disappearance containing toxin A gene, toxin B gene, binary toxin A gene (cdtA), binary toxin B gene (cdtB) and cdtC gene detect clostridium difficile, realize the Type of toxin of differentiating clostridium difficile.Specifically, the present invention is by adding the detection to tpi locus, can carry out specific detection to clostridium difficile, be conducive to prevent the erroneous judgement that traditional locus may occur in detecting, in addition, can detect to the detection of tcdC locus the gene whether bacterial strain in sample can contain C toxin by adding.
Above-mentioned primer is preferred and the preferred concentration of primer in amplification system is as shown in table 1:
Table 1 is primer sequence and concentration preferably
In corresponding 2 primers of above-mentioned each locus, have at least 1 to be through fluorescently-labeled, preferably adopt any one in FAM, HEX, TAMRA or ROX etc. to carry out mark.
The preferred amplification system of above-mentioned test kit is as shown in table 2.
Table 2PCR application of sample system
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 0.1～10 μ l content is 0.5pg～0.5ng |
| Primer mixture | 5μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
In wherein said Reaction Mix, contain MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.Wherein genome DNA sample can be bacterium liquid, can be also to extract DNA.
Another object of the present invention has been to provide a kind of fluorescent mark multiplex PCR clostridium difficile detection method based on mentioned reagent box, comprises the steps:
The 1st step, extraction target dna sample;
The amplification system of the 2nd step, employing test kit carries out multiplex PCR amplification to DNA sample;
The 3rd step, amplified production is carried out to electrophoresis, and result is analyzed.
Preferred Amplification in above-mentioned pcr amplification is as shown in table 3:
The amplification program of table 3 thermal cycler
Electrophoresis preferably adopts multiple tracks or single track capillary electrophoresis.
Beneficial effect
Beneficial effect of the present invention is: provide a kind of qualification clostridium difficile to have or not, and contratoxin A, toxin B, whether being always or usually as specified of the toxin such as binary toxin A and binary toxin lacks with tcdC gene the test kit and the analytical procedure that detect simultaneously.In once increasing, detecting, provide more information.Actual sample detection validation, the present invention is through lot of experiments and optimization, by the design to primer and the adjustment to amplification system and parameter, can be to being low to moderate the accurate somatotype of clostridium difficile of 26 copies.
Brief description of the drawings
Accompanying drawing 1a is that in embodiment 2, sample dilutes the amplification figure of 6 times, and the first row amplified peak is toxin amplified peak, is followed successively by the tcdC amplified peak that lacks 18bp, tpi gene amplification peak, tcdA gene amplification peak, cdtA gene amplification peak, tcdB amplified peak, cdtB amplified peak, amplification peak height is all more than 8000; The second row peak is the peak of molecular weight marker AGCU SIZ-500, is produced and is sold, with siz fluorescent mark, from 75bp to 500bp by Zhongde Meilian Biotech Co., Ltd. Wuxi, respectively 75,100,139,150,160,200,250,300,340,350,400,450,490,500, totally 14 fragments.
Accompanying drawing 1b is that in embodiment 2, sample dilutes the amplification figure of 17 times, lacks the tcdC of 18bp, tpi gene, and tcdA gene, cdtA gene, tcdB amplification, cdtB amplification has amplification, and peak height is in 7000 left and right.
Accompanying drawing 1c is that in embodiment 2, sample dilutes the amplification figure of 19 times, lacks the tcdC of 18bp, tpi gene, and tcdA gene, cdtA gene, tcdB amplification, cdtB amplification has amplification, and peak height is in 3000 left and right.Template concentrations is now 54.2fg/ μ l, 25 μ l amplification system application of sample 2 μ l, and in amplification system, template total amount is 108.4fg, is equivalent to 26 clostridium difficile genome copies.Accompanying drawing 1d is that in embodiment 2, sample dilutes the amplification figure of 20 times, lacks the tcdC of 18bp, tpi gene, and tcdA gene, cdtA gene, tcdB amplification, cdtB amplification has amplification, and peak height is in 1000 left and right.Except these peaks that should go out in theory, there is the amplified peak (being labeled as nor) not lacking in tcdC.Infer the minimizing along with template amount, the non-specific amplification that primer random amplification or amplification slippage cause.So far weaker concn, amplification starts the unreliable of change.
Accompanying drawing 1e is the amplification figure of sample dilution 21 times in embodiment 2, and two amplified peak appear in tcdC, the cdtB loss that increases, and all the other peak heights that increase are in 100 left and right.
Accompanying drawing 1f is that in embodiment 2, sample dilutes the amplification figure of 22 times, and except tpi, cdtA peak height are higher than 50, all the other gene amplification peak heights are all lower than 50.
Accompanying drawing 2a is the augmentation detection figure of a class sample in embodiment 3, and the first row peak is for detecting amplified peak, tcdA, and tcdB, tcdC, tpi, tcdA and tcdB be without amplification, the second behavior molecular weight marker SIZ500.
Accompanying drawing 2b is the augmentation detection figure of another kind of sample in embodiment 3, and the first row peak is for detecting amplified peak, and tpi has amplification, tcdA, and tcdB, tcdC, tcdA, tcdB is without amplification; The second behavior molecular weight marker, SIZ500.
Accompanying drawing 2c is the augmentation detection figure of another kind of sample in embodiment 3: the first row peak is detection amplified peak, tcdA, and tcdB, tcdC and tpi have amplification, and wherein tcdC is normal size, is labeled as NOR; TcdA, tcdB is without amplification; The second behavior molecular weight marker, SIZ500.
Accompanying drawing 2d is the augmentation detection figure of another kind of sample in embodiment 3: the first row peak is detection amplified peak, tpi, and tcdA, tcdB, tcdC, tcdA and tcdB have amplification, and wherein tcdC is the allelotrope level (being labeled as-1) of disappearance 18bp; The second behavior molecular weight marker, SIZ500.
Embodiment
By reference to the accompanying drawings the present invention is described in further detail below by embodiment.But it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (for example show with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or condition, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The design of embodiment 1 test kit
From NCBI genebank, download design of primers template sequence, as table 4.
Table 4 template sequence table
| Locus | No. Genebank, sequence | Position |
| tpi | AM180355.1 | 3706953-3707696 |
| tcdA | JQ809335.1 | 1-8133 |
| tcdB | AF217292.1 | 1-7512 |
| ctdC | HQ596359.1 | 1-1006 |
| cdtA | AF271719.1 | 1-1392 |
| cdtB | AF271719.1 | 1445-4075 |
Each locus sequence is compared in genebank, selects wherein the most conservative zone design primer.
Taking the design of primers of TcdA as example, design of primers template JQ809335.1 complete sequence, in NCBI blast comparison, has been determined to nt4000-6000 sequence-specific is higher, taking this part sequence as design of primers stencil design 4 pairs of primer sets, as following table.
The primer of table 5TcdA initial designs
primer is synthetic by the raw work in Shanghai, single primer is done to annealing temperature gradient amplification, the every pair of primer wherein one with FAM fluorescent mark.
The sample clostridium difficile strain 155 adopting is the bacterial strains that screened by Disease Control and Prevention Center of Zhejiang Province, and has determined whole genome sequence, and genome total length is 4.2MB, and single copy gene group weight is 4.2fg.Through the checking of order-checking row, this bacterial strain is containing binary toxin A and B, toxin A and toxin B and have tcdC18bp disappearance.6 genome sequences that the present invention relates on this bacterial strain and the homology of the disclosed sequence of genebank reach 100%.
The sample used that increases is that the every μ l of 28.4ng/ μ l(that Disease Control and Prevention Center of Zhejiang Province provides contains 6.8 × 10
6copy genomic dna) 155 bacterial strain DNA profilings.Amplification system while carrying out pcr amplification is as table 6.
Table 6PCR application of sample system
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 0.1～10 μ L content is 0.5pg～0.5ng |
| Primer mixture | 5μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
In wherein said Reaction Mix, contain MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.
Carry out again pcr amplification thermal cycling:
(l) pcr amplification pipe is placed on thermal cycler;
(2) program of option table 7 increases;
(3) sample after amplification should keep in Dark Place;
Table 7 thermograde amplification program
Wherein elongating temperature is respectively 54 DEG C, and 56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C, 64 DEG C.All the other step temperature are identical.Do gradient test.
, then carry out amplified production fluoroscopic examination on genetic analyzer next.
Form loading mixture ((0.5 μ lAGCU Marker SIZ-500 (marking in the siz500 fluorescent mark of preparation method with reference to our company's independent research disclosing in patent CN101307226, altogether 13 fluorescent mark fragment: 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 500bp)) × (sample introduction number)+(12 μ l deionized formamide) × (sample introduction number)) by marking AGCU Marker SIZ-500 in molecular weight in deionized formamide and system.12.5 μ l loading mixtures are mixed with PCR product, avoid producing bubble.95 DEG C of sex change 3 minutes, ice bath 3 minutes, and electrophoresis as early as possible; Detect and analyze with genetic analyzer; Detect the data of collecting with genetic analyzer in fragment analysis software GeneMapper analytical procedure C.Electrophoresis adopts multiple tracks or single track capillary electrophoresis.
Through experiment test, primer sets 3,4 is easily lost 62 DEG C, 64 DEG C amplifications; Primer sets 2 has non-specific amplification below at 56 DEG C; Primer sets 1 is low at part sample amplification efficiency, through sequence verification, finds that causing the reason of this problem is that 1R primer skims apart from 3 the sudden change that has t>c on the 3rd the corresponding sample sequence of base of end.
Grope through a large amount of tests, find to be that SEQ ID NO.3～4 overcome above-mentioned amplification problem preferably by primer 5(, as shown in table 8)
The primer of the preferred TcdA initial designs of table 8
Finally determine that primer 5 enters composite amplification test.Similarly, also need the corresponding primer of other locus to design and test, then test by composite amplification the adjustment again of carrying out design of primers, concentration and Amplification.
After readjusting through the sequence of a large amount of primer and concentration, finally determined primer sequence and primer concentration, as table 9, the application of sample system of composite amplification primer test is as table 10, and Amplification is as table 11.Primer amplification efficiency is high, each other without non-specific amplification.The size of amplified production is consistent with expection, after amplified production is cut to glue, checks order, consistent with target sequence.Verify the accuracy of this test kit.
Table 9 is primer sequence and concentration preferably
Table 10PCR application of sample system
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 0.1～10 μ l content is 0.5pg～0.5ng |
| Primer mixture | 5μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
The amplification program of table 11 thermal cycler
Embodiment 2 system sensitivity tests
Clostridium difficile strain 155 is the bacterial strains that screened by Disease Control and Prevention Center of Zhejiang Province, and has determined whole genome sequence, and genome total length is 4.2MB, and single copy gene group weight is 4.2fg.This bacterial strain is containing binary toxin A and B, toxin A and toxin B and have tcdC18bp disappearance.
The every μ l of 28.4ng/ μ l(that Disease Control and Prevention Center of Zhejiang Province is provided is containing 6.8 × 10
6copy genomic dna) 155 bacterial strain DNA, with doubling dilution dilution, serial dilution 22 times, the concentration after dilution is 14.2ng/ μ l (every microlitre copy number 3.4 × 10 for the first time
6); The concentration of the 22nd dilution is 6.8fg/ μ l, and every μ l is approximately containing 1.6 copy genomes.Taking the template after these 22 dilutions as amplification template, the detectability of detection system (sensitivity).
Primer adopts as shown in table 9, the every pair of primer wherein one with FAM fluorescent mark, by the application of sample system application of sample of table 12, increase by the amplification program of table 13.
Table 12PCR application of sample system
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 2μl |
| Primer mixture | 5μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
In wherein said Reaction Mix, contain MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.
Table 13PCR amplification program
Amplification is analyzed by the electrophoresis detection in embodiment 1, data analysing method, and each weaker concn repeats 3 pipe amplifications.
Along with increasing of extension rate, amplified peak progressively reduces.When the 20th dilution, there is non-specific amplification in tcdC, and when the 21st dilution, amplified peak height, in 200, occurs increasing and loses.Part Amplification Analysis result is as accompanying drawing 1a-1f.(accompanying drawing is the collection of illustrative plates of electrophoresis result after the genemapper IDX of American AB company software analysis.）
Accompanying drawing 1a dilutes the amplification of 6 times, and the first row amplified peak is toxin amplified peak, is followed successively by the tcdC amplified peak that lacks 18bp, tpi gene amplification peak, tcdA gene amplification peak, cdtA gene amplification peak, tcdB amplified peak, cdtB amplified peak, amplification peak height is all more than 8000; The second row peak is the peak of molecular weight marker AGCU SIZ-500, is produced and is sold, with siz fluorescent mark, from 75bp to 500bp by Zhongde Meilian Biotech Co., Ltd. Wuxi, respectively 75,100,139,150,160,200,250,300,340,350,400,450,490,500, totally 14 fragments.
Accompanying drawing 1b dilutes the amplification of 17 times, lacks the tcdC of 18bp, tpi gene, and tcdA gene, cdtA gene, tcdB amplification, cdtB amplification has amplification, and peak height is in 7000 left and right.
Accompanying drawing 1c dilutes the amplification of 19 times, lacks the tcdC of 18bp, tpi gene, and tcdA gene, cdtA gene, tcdB amplification, cdtB amplification has amplification, and peak height is in 3000 left and right.Template concentrations is now 54.2fg/ μ l, 25 μ l amplification system application of sample 2 μ l, and in amplification system, template total amount is 108.4fg, is equivalent to 26 clostridium difficile genome copies.
Accompanying drawing 1d dilutes the amplification of 20 times, lacks the tcdC of 18bp, tpi gene, and tcdA gene, cdtA gene, tcdB amplification, cdtB amplification has amplification, and peak height is in 1000 left and right.Except these peaks that should go out in theory, there is the amplified peak (being labeled as nor) not lacking in tcdC.Infer the minimizing along with template amount, the non-specific amplification that primer random amplification or amplification slippage cause.So far weaker concn, amplification starts the unreliable of change.
Accompanying drawing 1e is the dilution amplification of 21 times, and two amplified peak appear in tcdC, the cdtB loss that increases, and all the other peak heights that increase are in 100 left and right.
Accompanying drawing 1f dilutes the amplification of 22 times, and except tpi, cdtA peak height are higher than 50, all the other gene amplification peak heights are all lower than 50.
For ensureing the reliable of detected result, setting detection sensitivity is 25 μ l systems, and template 108.4fg is equivalent to 26 clostridium difficile genome copies.
Embodiment 3
13 fecal samples are so kind as to give by Zhejiang Disease Control and Prevention Center, in advance in Disease Control and Prevention Center of Zhejiang Province with spawn culture method, substance quantitative PCR detection, identification and analysis is crossed bacterial strain.
The genomic extraction of ight soil is the QIAamp DNA stool Mini Kit test kit with Qiagen company, undertaken by operation instructions, template through quantitative concentrations at 0.5～2ng/ μ l.
13 ight soil genomic dnas press embodiment 1 final determine primer sets, amplification system, amplification program amplification, and as the detection method detection of embodiment 1.Software analysis peak height, higher than 200rfu, thinks that corresponding gene has amplification.Otherwise analytical results peak height, lower than 200rfu, is thought without amplification, not containing corresponding gene.
13 samples, detect and are divided into Four types through this programme: 3 samples are without amplified peak, not containing clostridium difficile (augmentation detection figure is as accompanying drawing 2a); 4 samples only tpi have amplified peak, and toxin genes involved, without amplification, proves not containing toxin (amplification figure is as accompanying drawing 2b); 5 sample toxin As, toxin B, tpi has amplification, and tcdC has amplification and without base deletion, is the clostridium difficile containing toxin A, toxin B (amplification figure is as accompanying drawing 2c); 1 sample toxin A, toxin B, binary toxin A, binary toxin B, tpi has amplification, and tcdC has amplification and has 18 base deletions (amplification figure is as 2d).The detected result of four kinds of types is as accompanying drawing 2a～2d, without specific amplification.Detected result and spawn culture, substance quantitative PCR detection result are consistent, carry out sequence verification after amplified production is cut to glue, reach 100% with the homology of open sequence.
SEQUENCE LISTING
<110> Zhongde Meilian Biotech Co., Ltd. Wuxi
<120> fluorescent mark multiplex PCR clostridium difficile detection kit
<130> without
<160> 20
<170> PatentIn version 3.5
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Claims (8)
1. a fluorescent mark multiplex PCR clostridium difficile detection kit, is characterized in that, includes the primer for the following locus of the clostridium difficile that increases: tpi, tcdA, tcd B, tcdC, cdtA and cdtB.
2. fluorescent mark multiplex PCR clostridium difficile detection kit according to claim 1, is characterized in that, described primer sequence is: tpi, SEQ ID NO.1～2; TcdA, SEQ ID NO.3～4; TcdB, SEQ ID NO.5～6; TcdC, SEQ ID NO.7～8; CdtA, SEQ ID NO.9～10; CdtB, SEQ ID NO.11～12.
3. fluorescent mark multiplex PCR clostridium difficile detection kit according to claim 2, is characterized in that, the concentration of each primer in amplification system is: SEQ ID NO.1～2,0.80 μ M; SEQ ID NO.3～4,0.60 μ M; SEQ ID NO.5～6,0.50 μ M; SEQ ID NO.7～8,1.0 μ M; SEQ ID NO.9～10,1.1 μ M; SEQ ID NO.11～12,0.85 μ M.
4. fluorescent mark multiplex PCR clostridium difficile detection kit according to claim 1, is characterized in that: in corresponding 2 primers of each locus, have at least 1 to be through fluorescently-labeled.
5. fluorescent mark multiplex PCR clostridium difficile detection kit according to claim 1, it is characterized in that: in corresponding 2 primers of each locus, having at least 1 to be through fluorescently-labeled, is to adopt any one in FAM, HEX, TAMRA or ROX to carry out mark.
6. fluorescent mark multiplex PCR clostridium difficile detection kit according to claim 3, it is characterized in that test kit composed as follows: Reaction Mix 10.0 μ L, genomic dna 0.1～10 μ l, primer mixture 5 μ L, warm start Taq enzyme 0.5 μ L, sdH
2o complements to 25.0 μ L; In described Reaction Mix, contain MgCl
27.5mM, Tris-HCl buffer 125mM, KCl 125mM, dNTPs 7.5mM, BSA 2mg/ml.
7. a fluorescent mark multiplex PCR clostridium difficile detection method for the test kit based on described in claim 1～6 any one, comprises the steps: the 1st step, extracts target dna sample; The amplification system of the 2nd step, employing test kit carries out multiplex PCR amplification to DNA sample; The 3rd step, amplified production is carried out to electrophoresis, and result is analyzed.
8. fluorescent mark multiplex PCR clostridium difficile detection method according to claim 7, is characterized in that, the Amplification in amplification is: initial sex change: 95 DEG C 2 minutes; Thermal cycling, 94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulation; Extend eventually, 72 DEG C, 10 minutes.
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| CN105039516A (en) * | 2015-06-04 | 2015-11-11 | 广州市第一人民医院 | Fluorescent PCR detection method for clostridium difficile toxin genes, as well as primer and kit of fluorescent PCR detection method |
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| CN110055311A (en) * | 2019-04-17 | 2019-07-26 | 江苏佰思瑞生物科技有限公司 | Clostridium difficile fluorescence quantitative PCR detection primer sets and kit |
| CN112481395A (en) * | 2019-09-12 | 2021-03-12 | 深圳华大生命科学研究院 | Clostridium difficile drug-resistant/low-sensitive clade SNP marker, strain type identification method and application |
| CN111826453A (en) * | 2020-08-06 | 2020-10-27 | 上海交通大学 | Clostridium difficile detection primer or dual detection primer group, kit and rapid detection method thereof |
| CN111876506A (en) * | 2020-08-17 | 2020-11-03 | 中国疾病预防控制中心传染病预防控制所 | Multiple fluorescent quantitative PCR primer, probe and kit for detecting toxigenic clostridium difficile |
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