CN110055311A - Clostridium difficile fluorescence quantitative PCR detection primer sets and kit - Google Patents
Clostridium difficile fluorescence quantitative PCR detection primer sets and kit Download PDFInfo
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- CN110055311A CN110055311A CN201910307822.7A CN201910307822A CN110055311A CN 110055311 A CN110055311 A CN 110055311A CN 201910307822 A CN201910307822 A CN 201910307822A CN 110055311 A CN110055311 A CN 110055311A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses a kind of clostridium difficile fluorescence quantitative PCR detection primer sets and its kit, the primer sets are made of TcdA primer, TcdB primer and GS primer；TcdA primer includes nucleotide sequence successively TcdA upstream primer, TcdA downstream primer and TcdA probe as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3；TcdB primer includes nucleotide sequence successively TcdB upstream primer, TcdB downstream primer and TcdB probe as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6；GS primer includes nucleotide sequence successively GS upstream primer, GS downstream primer and GS probe as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9；5 ' ends of TcdA probe, TcdB probe and GS probe nucleotide sequence are connect with fluorophor, and 3 ' ends are connect with quencher, and TcdA probe, TcdB probe and the 5 ' ends of GS probe three are separately connected different fluorophors.
Description
Technical field
Microorganism field of the present invention, especially a kind of primer sets and reagent for clostridium difficile fluorescence quantitative PCR detection
Box.
Background technique
Clostridium difficile (Clostridium difficile) is also known as clostridium difficile, difficulty distinguishes bacteroides fusiformis or difficult shuttle
Bacterium belongs to Anaerobic Bacteria, has been acknowledged as causing antibiotic-associated diarrhea (AAD) and pseudomembranous enteritis (PMC) at present
Important pathogenic bacteria.In addition to above-mentioned disease, clostridium difficile still causes pyelonephritis, meningitis, abdominal cavity and vagina infection, bacteremia
With emphysematous gangrene etc., the bacterium has become one of pathogen of inside-hospital infection in recent years.Cause the difficult shuttle of production poison of clinical symptoms
Bacterium, toxin producing type includes toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA/cdtB), wherein the toxin on basis
The factor is toxin A and toxin B.Country's clinical diagnosis clostridium difficile toxin mainly uses Enzyme-linked Immunosorbent Assay (ELISA) to examine at present
Toxin B is surveyed, or is identified after carrying out Anaerobic culturel to doubtful sample, this method detection time is long, operates comparatively laborious, Er Qiejian
It is small to survey flux, is not suitable for large-scale screening.Further, since the sample of detection is that excrement is easy to make containing more interfering substance
At false negative etc., the accuracy of detection is influenced.And the operation identified for the Anaerobic culturel of doubtful sample is more cumbersome, must have
Perfect experiment condition and more demanding to the experimental level of detection, is equally not suitable for extensive screening.
The application of fluorescence quantitative PCR method detection pathogen is increasingly closed by hospital laboratory because of the advantages that its is quick, sensitive
Note, corresponding product are also just updating rapidly, substitute original out of season detection method.Medical University Of Fujian master opinion
" multiple PCR method detection Clostridium difficile toxin A gene, toxin b gene and binary toxin gene, Pan Shaomin " disclose one kind to text
The method of clostridium difficile is detected using PCR and discloses relevant primer, and the method that the document uses regular-PCR needs
Photographic analysis under agarose gel electrophoresis and ultraviolet gel image analyser is carried out, the time is expended, trivial operations, and agarose
It is strong mutagens that nucleic acid staining reagent ethidium bromide (EB) is commonly used in gel electrophoresis, has potential carcinogenic risk, influences to operate
Person's health；There is not been reported for the current primer for using fluorescence quantitative PCR method detection for clostridium difficile and kit.
Summary of the invention
Technical problem to be solved by the present invention lies in prior art defect is overcome, provides and a kind of designed according to target sequence
The primer sets of specificity, the difficile dna target fragment of toxin producing is expanded, is detected by multiple fluorescence quantitative PCR
The detection primer group and kit of Clostridium difficile toxin A (tcdA) and toxin B (tcdB).
To achieve the above object, the invention is realized by the following technical scheme:
Present invention firstly provides a kind of clostridium difficile fluorescence quantitative PCR detection primer sets, the primer sets include TcdA primer,
TcdB primer and GS primer；The TcdA primer includes nucleotide sequence successively such as SEQ ID NO.1, SEQ ID NO.2 and SEQ
TcdA upstream primer, TcdA downstream primer shown in ID NO.3 and TcdA probe；The TcdB primer includes nucleotides sequence Leie
Secondary TcdB upstream primer, TcdB downstream primer and TcdB as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6
Probe；The GS primer includes nucleotide sequence successively as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9
GS upstream primer, GS downstream primer and GS probe；5 ' ends of the TcdA probe, TcdB probe and GS probe nucleotide sequence are equal
It is connect with fluorophor, 3 ' ends are connect with quencher, and TcdA probe, TcdB probe and the 5 ' ends point of GS probe three
Different fluorophors is not connected；
Above-mentioned fluorophor and quencher are the fluorescent dye groups of this field routine, and the fluorophor includes but unlimited
In groups such as FAM, VIC, ROX, CY5, JOE, quencher includes but is not limited to the groups such as TRAMA, BHQ1, BHQ2.
Further, in clostridium difficile fluorescence quantitative PCR detection primer sets provided by the invention, 5 ' ends of the TcdA probe
FAM group and BHQ1 group are separately connected with 3 ' ends；5 ' the ends and 3 ' ends of the TcdB probe are separately connected ROX group and BHQ2
Group；5 ' the ends and 3 ' ends of the GS probe are separately connected JOE group and BHQ1 group.
Secondly, the present invention also provides a kind of for detecting the kit of clostridium difficile, which is characterized in that the kit
In contain primer sets as claimed in claim 1 or 2.
Further, provided by the present invention for the kit of detection clostridium difficile, comprising: under TcdA upstream primer, TcdA
Primer, TcdA probe, TcdB upstream primer, TcdB downstream primer, TcdB probe, GS upstream primer, GS downstream primer, GS is swum to visit
Needle.
Third, the present invention also provides a kind of methods of fluorogenic quantitative detection clostridium difficile, the specific steps of which are as follows:
1) sample DNA is extracted
According to this field routine difference DNA extraction method or select product " faeces DNA genome extraction kit " on the market
The DNA that by specification operation carries out sample excrement is extracted.
2) three kinds of primers and probe are prepared
1 three kinds of primers of table and probe
| TcdA upstream primer | AGATTCCTATATTTACATGACAATAT |
| TcdA downstream primer | GTATCAGGCATAAAGTAATATACTTT |
| TcdA probe | FAM-AGATATTACTTCGAGCCTAATACAGC-BHQ1 |
| TcdB upstream primer | GGAAAAGAGAATGGTTTTATTAA |
| TcdB downstream primer | ATCTTTAGTTATAACTTTGACATCTTT |
| TcdB probe | ROX-CTGATGTAGTTCTTATAAGTAA-BHQ2 |
| GS upstream primer | GATCCCGATAGGCTCCACCACTTT |
| GS downstream primer | ATCCACCGTGCGCCCTTTCTAGCT |
| GS probe | JOE-ACCGGAAAAGCGGAGGCGAGTGTT-BHQ1 |
1 middle probe of table selects suitable fluorophor and quenches respectively by the fluorochrome label of three kinds of different sense channels
It goes out moiety combinations.
If primer and probe are when carrying out nucleic acid extraction, the exogenous DNA fragmentation of addition is used for GS (internal reference) in table 1
Whether evaluation nucleic acid extraction kit operation there is exception.
3) reagent prepares to prepare PCR reaction buffering by material supplier's quantitative fluorescent PCR (sonde method) kit specification
Liquid, (4000rpm centrifugation 10s processing after all reagents are vortexed before configuration)；
2 PCR reaction premixed liquid of table matches table
| Reagent | Volume |
| PCR Buffer | 35μL |
| Primer Mix | 4.7μL |
| DNA Taq polymerase | 0.3μL |
| Total volume | 40.0μL |
In table 2, Primer Mix includes: that 0.6 μ L of TcdA upstream primer that concentration is 10 μM, the downstream TcdA that concentration is 10 μM are drawn
0.6 μ L of object, 0.6 μ L of TcdB upstream primer that concentration is 10 μM, 0.6 μ L of TcdB downstream primer that concentration is 10 μM, concentration are 10 μ
0.6 μ L of GS upstream primer of M, 0.6 μ L of GS downstream primer that concentration is 10 μM, 0.4 μ L of TcdA probe that concentration is 10 μM, concentration
For 10 μM of 0.4 μ L of TcdB probe, the 0.4 μ L of GS probe that concentration is 10 μM.
4) step 1) is obtained fecal sample DNA10 μ L (concentration is about 0.1ng/ μ L) and added respectively by fluorescent PCR augmentation detection
Into the PCR reaction tube equipped with PCR premixed liquid, the 4000rpm after mixing that is vortexed is centrifuged 10s.
PCR reaction tube is put into fluorescent PCR amplification instrument and carries out augmentation detection；
PCR amplification program is as follows: 95 DEG C of processing 3min；95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s (collecting fluorescence signal), 40 are followed
Ring；
Instrument channel selection: the channel toxin A-FAM；The channel toxin B-ROX；The channel internal reference-JOE (no ROX fluorescent dye correction).
Above procedure is run, after the completion of amplification, if there is " s " type increased logarithmic phase amplification curve, then it is assumed that sample contains
Toxin A perhaps toxin B (positive) conversely, then judgement sample be free of toxin A or toxin B (feminine gender).
In the application, positive sample refers to the fecal sample containing toxin A or toxin B, and negative sample refers to not toxic
The fecal sample of plain A or toxin B.
Compared with the existing detection method, the present invention provides method (Taqman sonde method) identifications for being quantitative fluorescent PCR
Primer sets and kit, it can testing result is directly obtained, quantitative analysis can also be carried out by standard curve, it is quick, quasi-
Really, high sensitivity, application easy to spread.
Detailed description of the invention
Fig. 1 is positive sample fluorescent quantitative PCR curve；
Fig. 2 is negative sample fluorescent quantitative PCR curve；
Fig. 3 is toxin A sensitivity test fluorescent quantitative PCR curve；
Fig. 4 is toxin B sensitivity test fluorescent quantitative PCR curve.
Specific embodiment
It elaborates below in conjunction with attached drawing to the present invention.
Faeces DNA genome extraction kit is purchased from Guangzhou Mei Ji Biotechnology Co., Ltd.
PCR (sonde method) kit is purchased from precious bioengineering (Dalian) Co., Ltd.
Positive sample is the fecal sample containing TcdA/TcdB in embodiment, to be originated from Chizhou City, Anhui Province the People's Hospital.
Negative sample is from Chizhou City, Anhui Province the People's Hospital through detecting the fecal sample of TcdA/TcdB feminine gender
The detection test of 1 clostridium difficile multiple fluorescence quantitative PCR of embodiment
1) the DNA extraction for carrying out sample excrement is operated according to " faeces DNA genome extraction kit " by specification；
2) three kinds of primers and probe (as shown in Table 1) are prepared
TcdA primer: TcdA upstream primer nucleotide sequence is as shown in SEQ ID NO.1, and TcdA downstream primer nucleotide sequence is such as
Shown in SEQ ID NO.2, TcdA probe nucleotide sequence is as shown in SEQ ID NO.3；Wherein, 5 ' and 3 ' ends point of TcdA probe
It Lian Jie not fluorophor FAM and quencher BHQ1；
TcdB primer: TcdB upstream primer nucleotide sequence is as shown in SEQ ID NO.4, and TcdB downstream primer nucleotide sequence is such as
Shown in SEQ ID NO.5, TcdB probe nucleotide sequence is as shown in SEQ ID NO.6；Wherein, 5 ' and 3 ' ends point of TcdB probe
It Lian Jie not fluorophor ROX and quencher BHQ2；
GS primer: GS upstream primer nucleotide sequence is as shown in SEQ ID NO.7, GS downstream primer nucleotide sequence such as SEQ ID
Shown in NO.8, GS probe nucleotide sequence is as shown in SEQ ID NO.9；Wherein, 5 ' and 3 ' ends of GS probe are separately connected fluorescence
Group JOE and quencher BHQ1.
3) reagent prepares
PCR reaction buffer is prepared by quantitative fluorescent PCR (sonde method) kit specification, (is carried out all reagents before configuration
Be vortexed centrifugation several seconds processing after mixing))
PCR reaction system: 4.7 μ L of Primer Mix, 0.3 μ L of DNA Taq polymerase, PCR Buffer complement to 40.0 μ L；
Primer Mix includes: 0.6 μ L of TcdA upstream primer that concentration is 10 μM, 0.6 μ L of TcdA downstream primer that concentration is 10 μM, dense
TcdB upstream primer 0.6 μ L, TcdB downstream primer 0.6 μ L that concentration be 10 μM, GS upstream that concentration be 10 μM of the degree for 10 μM
0.6 μ L of primer, 0.6 μ L of GS downstream primer that concentration is 10 μM, 0.4 μ L of TcdA probe that concentration is 10 μM, concentration are 10 μM
0.4 μ L of TcdB probe, the 0.4 μ L of GS probe that concentration is 10 μM.
4) fluorescent PCR augmentation detection
Will test sample: positive sample (containing toxin A and toxin B) and each 10 μ L of negative sample are added to premix equipped with PCR respectively
In the PCR reaction tube of liquid, the 4000rpm after mixing that is vortexed is centrifuged 10s.
The PCR reaction tube for the PCR reaction system prepared containing step 3) is put into fluorescent PCR amplification instrument and carries out amplification inspection
It surveys；
PCR amplification program is as follows: 95 DEG C of processing 3min；95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s (collecting fluorescence signal), 40 are followed
Ring；
Instrument channel selection: the channel toxin A-FAM；The channel toxin B-ROX；The channel internal reference-JOE (no ROX fluorescent dye correction)
The fluorescent quantitative PCR curve of positive sample and negative sample difference is as shown in Figure 1 and Figure 2 in the present embodiment, it is seen then that
There is " S " type increased logarithmic phase amplification curve, Fig. 2 feminine gender sample in Fig. 1 positive sample (containing toxin A or toxin B) testing result
This does not occur above-mentioned curve.The present embodiment detection method can intuitively, effectively detect negative sample and positive sample, and can
With the content of toxin A and toxin B in real-time detection positive sample.
The experiment of 2 sensitivity technique of embodiment
Detection sample in step 4) is only toxin A positive sample, the positive sample with embodiment 1 by detection method in the present embodiment
This preparation method is as follows: being successively diluted to 4.6 × 10 by concentration gradient (with the dilution of TE solution)6、4.6×105、4.6×104、
4.6×103、4.6×102、4.6×101(copies/test)。
Testing result is as shown in Figure 3, it is seen then that the application primer and kit detection TcdA sensitivity be 4.6 ×
102copies。
The experiment of 3 sensitivity technique of embodiment
Detection sample in step 4) is only toxin B positive sample, the positive sample with embodiment 1 by detection method in the present embodiment
This preparation method is as follows: being successively diluted to 4.6 × 10 (with the dilution of TE solution) by concentration gradient6、4.6×105、4.6×104、
4.6×103、4.6×102、4.6×101(copies/test)。
Testing result is as shown in Figure 4, it is seen then that the application primer and kit detection TcdB sensitivity be 4.6 ×
102copies。
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, several improvement can also be made without departing from the principle of the present invention, these improvement also should be regarded as of the invention
Protection scope.
Claims (4)
1. a kind of clostridium difficile fluorescence quantitative PCR detection primer sets, the primer sets are by TcdA primer, TcdB primer and GS primer
Composition；The TcdA primer includes nucleotide sequence successively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3
TcdA upstream primer, TcdA downstream primer and TcdA probe；The TcdB primer includes nucleotide sequence successively such as SEQ ID
TcdB upstream primer, TcdB downstream primer and TcdB probe shown in NO.4, SEQ ID NO.5 and SEQ ID NO.6；The GS
Primer include nucleotide sequence successively the GS upstream primer as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9,
GS downstream primer and GS probe；The TcdA probe, TcdB probe and GS probe nucleotide sequence 5 ' ends and fluorophor
3 ' ends of connection, the TcdA probe, TcdB probe and GS probe nucleotide sequence are connect with quencher, and TcdA is visited
Needle, the fluorophor that the 5 ' ends of TcdB probe and GS probe three are connected are all different.
2. clostridium difficile fluorescence quantitative PCR detection primer sets as described in claim 1, which is characterized in that the fluorophor
Including FAM, VIC, ROX, CY5, JOE group, the quencher includes TRAMA, BHQ1, BHQ2 group.
3. clostridium difficile fluorescence quantitative PCR detection primer sets as claimed in claim 2, which is characterized in that the TcdA probe
5 ' end and 3 ' end be separately connected FAM group and BHQ1 group；5 ' the ends and 3 ' ends of the TcdB probe are separately connected ROX group
With BHQ2 group；5 ' the ends and 3 ' ends of the GS probe are separately connected JOE group and BHQ1 group.
4. a kind of for detecting the kit of clostridium difficile, which is characterized in that appoint in the kit containing such as claim 1-3
Primer sets described in one.
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| WO2013031973A1 (en) * | 2011-09-01 | 2013-03-07 | 株式会社ヤクルト本社 | Method for detecting toxin-producing clostridium difficile |
| CN103361434A (en) * | 2013-07-24 | 2013-10-23 | 浙江省疾病预防控制中心 | Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes |
| CN103911430A (en) * | 2014-02-21 | 2014-07-09 | 无锡中德美联生物技术有限公司 | Fluorescence labeling multiplex PCR detection kit for Clostridium difficile |
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| CN105525023A (en) * | 2016-02-04 | 2016-04-27 | 广州赛哲生物科技股份有限公司 | Fluorescent quantitative PCR detection kit for clostridium difficile toxin A/B and detection method |
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2019
- 2019-04-17 CN CN201910307822.7A patent/CN110055311A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013031973A1 (en) * | 2011-09-01 | 2013-03-07 | 株式会社ヤクルト本社 | Method for detecting toxin-producing clostridium difficile |
| CN102952886A (en) * | 2012-11-28 | 2013-03-06 | 中华人民共和国张家港出入境检验检疫局 | Dual fluorescence quantitative PCR (polymerase chain reaction) detection method and detection kit for clostridium difficile enterotoxin A and B |
| CN103361434A (en) * | 2013-07-24 | 2013-10-23 | 浙江省疾病预防控制中心 | Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes |
| CN103911430A (en) * | 2014-02-21 | 2014-07-09 | 无锡中德美联生物技术有限公司 | Fluorescence labeling multiplex PCR detection kit for Clostridium difficile |
| 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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