CN103952496A - Duplex PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus - Google Patents
Duplex PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and concretely relates to a duplex PCR method for detecting porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The method comprises: designing two primers respectively amplifying the specific sequences of TGEV and PEDV, extracting RNA from positive intestinal canal tissue infected TGEV and PEDV, performing inverse transcription to obtain cDNA, and further taking cDNA as a template, so as to establish a multiplex PCR detection method capable of directly detecting two kinds of diarrhoea viruses from a sample once. The beneficial effects comprise that compared with presently reported PCR detection methods, the duplex PCR detection method is improved in detection efficiency and strong in specificity.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of multi-PCR detection method, particularly a kind of double PCR method that detects transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus in porcine tissue.
Background technology
Transmissible gastroenteritis of swine (transmissible gastroenteritis, TGE) be Transmissible gastroenteritis virus (the transmissible gastroenteritis virus by coronaviridae, TGEV) a kind of contagious disease of the pig causing, be worldwide distribution, from phase late 1960s, just there is the report about this disease in China, and epidemic-stricken area more expands in recent years, causes tremendous economic loss to pig industry.It is clinical taking vomiting, severe diarrhea and dehydration property enteritis as principal character.The equal susceptible of pig of different ages and kind, especially to 2 week age with interior piglet, its lethality rate is up to 100%.TGE virus belongs to coronaviridae coronavirus genus.
Porcine epizootic diarrhea (Porcine epidemic diarrhea, PED) be Porcine epidemic diarrhea virus (the porcine epidemic diarrhea virus by coronaviridae coronavirus genus, PEDV) pig causing acute, height contact infectious intestinal disease, China just had the report of PED successively since 1976, the popular of PED was on the rise in recent years, since second half 2010, In South China, east and North China part province has occurred that serious grice diarrhoea is popular, cause the death of a large amount of sucking pigletss, also caused huge financial loss to pig industry.It is clinical taking watery diarrhea, vomiting, dehydration as principal character, and pathological change is mainly manifested in the jejunum of pig and the intestinal villus atrophy of ileal segment and comes off.
Above two kinds of diseases are all the common viral diarrheas in pig farm, and viral diarrhea has caused the heavy losses such as pig farm price of deed reduction, medicine and manpower increase, are one of the most serious diseases in harm pig farm.Because TGE, PED have similar clinical manifestation and the route of infection, its differential diagnosis conventionally need be by laboratory diagnosis technology, as virus separation, immunofluorescent test, round pcr etc.Traditional diagnostic techniques wastes time and energy as virus separation, immunofluorescent test etc., is unsuitable for clinical quick diagnosis, is also unsuitable for large-scale epidemiology survey.And round pcr has high specificity, susceptibility is high, can carry out the features such as live body diagnosis, has more outstanding advantage in laboratory diagnosis.
Multiplex PCR (multiplex PCR) is improved on the basis of regular-PCR, adopts multipair Auele Specific Primer to the increase round pcr of multiple object fragments of the different zones of multiple DNA profilings or same template.It carries out pcr amplification by the Auele Specific Primer that adds multiple pathogenic microorganisms in same PCR reaction tubes simultaneously, and can detect multiple pathogens simultaneously or identify is that type pathogenic infection.
Due to multiplex PCR multiple goal gene that increase simultaneously, have advantages of and save time, reduce costs, raise the efficiency, particularly save precious laboratory sample, so once proposition, obtain numerous investigators' favor, and rapidly, in the every field of life science, multiplex PCR has become a maturation and important research means in development.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of directly multi-PCR detection method of one-time detection transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus from sample is provided, the PCR detection method of its more existing report has improved detection efficiency.
For two kinds of virus polyinfections often in actual production, very indistinguishable situation, the present invention utilizes information biology means, from affecting the various factors analyzing gene fragment structure of PCR detection sensitivity, filter out the gene segment the most easily going out by pcr amplification and detect target spot as PCR, analyze the primer of these target spot genes of design amplification from primer mass parameter aspect; Two couple who further utilizes design is to increase the respectively specific sequence of TGEV, PEDV of primer, with TGEV and (or) the doubtful infection sample extraction of PEDV RNA, further send out and be transcribed into cDNA, taking cDNA as amplification template, set up the double PCR system that transmissible gastroenteritis of swine, porcine epizootic diarrhea are infected to differential diagnosis.Its concrete technical scheme is described below.
The double PCR method that the invention provides a kind of Transmissible gastroenteritis virus and Porcine epidemic diarrhea virus, concrete steps comprise:
(1) from infecting the positive intestinal tube tissue extraction RNA of TGEV, PEDV, reverse transcription becomes cDNA, further taking cDNA as masterplate, 2 pairs of primers for TGEV and PEDV is respectively added to PCR reaction system simultaneously, carries out pcr amplification;
(2) after PCR finishes, PCR product is done to agarose gel electrophoresis analysis, obtain 385bp specific gene fragment and represent infected pigs's Transmissible gastroenteritis virus, obtain 628bp specific gene fragment and represent infected pigs's epidemic virus; Wherein:
In step (1), the sequence of 2 pairs of primers is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 and SEQ ID NO.4:
Upstream primer TGEV:5 ' CAACCCCGATGGAGCACT3 ';
Downstream primer TGEV:5 ' CCGAA CATTA CATAT CTGGA CACT3 ';
Upstream primer PEDV:5 ' TGAGG GTGACAAGTTCGTAGG3 ';
Downstream primer PEDV:5 ' CGCCA AGATATTAAAAGATTCACC3 ';
In above-mentioned steps (1), PCR reaction system is as follows: cDNA template 1 μ l, and 2 couples of each 0.5 μ l of upstream and downstream primer, PCR Mix12.5 μ l, ultrapure water is supplemented to 50 μ l; Pcr amplification reaction condition is as follows: 95 DEG C of denaturation 5min, enter 95 DEG C of 60s, and the circulation of 52 DEG C of 1min and 72 DEG C of 1min, circulates 35 times, and last 72 DEG C are extended 5min.
Compared with prior art, the present invention utilizes the gene fragment oneself screening to detect target spot as ideal, explore at aspects such as annealing temperature, primer and other reaction system parameters, filter out multiplex PCR detection system highly sensitive, high specificity, set up the directly multi-PCR detection method of two kinds of diarrhea viruses of one-time detection from sample, experiment showed, the PCR detection method of more existing report, multi-PCR detection method of the present invention is reproducible, high specificity, detection efficiency are high.
Brief description of the drawings
Fig. 1 is the single pcr amplification result figure that comparative example 1 is tested sample.M is standard molecular weight sample, and 1,2 is two doubtful infection PEDV viral sample, consistent with the object fragment (628bp) of prediction; 3,4 negative contrasts.
Fig. 2 is the single pcr amplification result figure that comparative example 1 is tested sample.M is standard molecular weight sample, and P4 is TGEV virus infection sample, consistent with the object fragment (385bp) of prediction; The negative contrast of P3.
Fig. 3 is that the different test samples of embodiment 1 carry out multiple PCR primer specific amplification result figure.M is standard molecular weight sample, and 1,2 is that two viruses infect sample simultaneously, and 3,4 infect separately sample for TGEV.
Fig. 4 is multiple PCR primer specific amplification result figure in embodiment 2.M is standard molecular weight sample, and 1,2 for doubtful two TGEV and PEDV infect sample simultaneously, and 3 is PRRSV, and 4 is CSFV, 5PRAV.
Embodiment
The present embodiment is implemented under taking technical solution of the present invention as prerequisite, has provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to reagent manufacturer.
The present invention utilizes a kind of information biology means, and first filter out virus (TGEV and the PEDV) gene segment the most easily going out by pcr amplification and detect target spot as PCR, and then the primer of these target spot genes of design amplification, its method is specific as follows:
1) the complete sequence screening of virus (TGEV and PEDV) testing goal fragment
By website
www.ncbi.nlm.nih.gov/Genbank, two goal gene that first input will be looked for respectively in Nucleotide mono-hurdle of homepage, the complete genome sequence being had from database afterwards downloads.
Porcine?epidemic?diarrhea?virus，complete?genome
27，953bp?Iinear?RNA
Accession：KC189944.1?GI：509265008
GenBank?
FASTA?
Granhics
TGEV?virulent?Purdue，complete?genome
28，544bp?Iinear?RNA
Accession：DQ811789.2GI:331265425
GenBank?
FASTA?
Granhics?
Related?
Sequences
2) design of primers of virus (TGEV and PEDV) tested gene target spot
The document of the complete sequence by above-mentioned these two virogenes of finding after order-checking is opened by DNAstar software, according to DNAstar working instructions, after sequence is read, according to setting requirement, (dimer hairpin structure is no more than 1 to software, annealing temperature is between 48 to 55 degree) screen, the primer scheme again gene order being provided by software is optimized and mates, thereby designs the primer of the tested gene target spot of virus (TGEV and PEDV); The sequence of 2 pairs of primers is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 and SEQ ID NO.4:
Upstream primer TGEV:5 ' CAACCCCGATGGAGCACT3 ';
Downstream primer TGEV:5 ' CCGAA CATTA CATAT CTGGA CACT3 ';
Upstream primer PEDV:5 ' TGAGG GTGACAAGTTCGTAGG3 ';
Downstream primer PEDV:5 ' CGCCA AGATATTAAAAGATTCACC3 ';
Wherein the specific gene fragment of transmissible gastro-enteritis virus is 385bp, and Porcine epidemic diarrhea virus is 628bp.
Comparative example 1
1RNA extracts the present invention RNA used Trizol method and extracts.Method reference reagent box specification sheets.The first step take certain pig farm doubtful infection TGEV or (with) PEDV pig intestinal tissue 20g, grind, add Trizol Reagent200 μ L, 5min is hatched in room temperature effect, makes its abundant cracking.The centrifugal 5min of 12000r/min, discards precipitation.Second step adds 200 μ L chloroforms, thermal agitation 15s, and room temperature leaves standstill 15min, and 4 DEG C, the centrifugal 15min of 12000r/min, draws upper strata water, to another centrifuge tube.The 3rd step adds 500 μ l Virahols, 1mlTrizol precipitated rna, and room temperature leaves standstill 10min; 4 DEG C, the centrifugal 10min of 12000r/min, abandons supernatant, and RNA is sunken to the pipe end.The 4th step, adds 1mL75% ethanol, 1mlTrizol, gentle vibration centrifuge tube, the precipitation that suspends, washing RNA precipitation.The 5th step, 4 DEG C, the centrifugal 5min of 8000r/min.Abandon supernatant, room temperature is dried or vacuum-drying 5-10min as far as possible, makes to be fully dried, and generally can see that facing to light some transparent materials are at the pipe end.Add 16 μ L DEPC water dissolution.DEPC water can deactivation range protein, is the potent inhibitor of RNA enzyme, can be used for RNA resolution of precipitate.
2cDNA is synthetic
Taking RNA as template, synthetic cDNA chain.The first step adds 8 μ lRNA templates and 2 μ l PrimeScript in 1.5mL centrifuge tube
tM(ThermoScript II) RT Master Mix (Perfect Real Time).37 DEG C of 15min of reaction conditions, 85 DEG C of 5s, 4 DEG C of 5min, reaction finishes rear ice bath and preserves.Obtain cDNA.
3 single pathogen gene pcr amplifications
Taking the synthetic cDNA of step 2 as template, respectively taking sequence as SEQ ID NO.1, SEQ ID NO.2 and 2 pairs of primers of SEQ ID NO.3, SEQ ID NO.4 do single amplification to primer.Amplification system is the each 0.5 μ l of upstream and downstream primer, and ultrapure water 10.5 μ l, PCR Mix(Shanghai Sheng Gong biotechnology company limited purchases) 12.5 μ l, cDNA template 1 μ l, has the mixture of 25 μ l in single system.95 DEG C of denaturation 5min, then continue 95 DEG C of 60s, and the annealing temperature of the two is respectively TGEV52.9 DEG C, PEDV54.2 DEG C, therefore annealing temperature is set to 52 degrees Celsius, after enter 72 DEG C of 60s circulation, circulate altogether 30 times, last 72 DEG C extend 5min.Preserve PCR product for 4 DEG C.After PCR finishes, get 8 μ l products and do agarose gel electrophoresis analysis, result respectively as shown in Figure 1 and Figure 2.
Embodiment 1
1RNA extracts
Method is with comparative example 1.
2cDNA is synthetic
Method is with comparative example 1.
The foundation of 3 multiplex PCRs
With doubtful infection TGEV or (with) the sample extraction RNA of PEDV, transcribe and obtain cDNA, taking this cDNA as template, 2 pairs of primers that sequence are respectively to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, SEQ ID NO.4 add amplification system simultaneously, set up double pcr amplification reaction system.Double PCR reaction system is as follows: cDNA template 1 μ l, and 2 couples of each 0.5 μ l of upstream and downstream primer, PCR Mix12.5 μ l, ultrapure water is supplemented to 50 μ l.PCR reaction conditions: 95 DEG C of denaturation 5min, enter 95 DEG C of 60s, the circulation of 52 DEG C of 1min and 72 DEG C of 1min, circulates 35 times, and last 72 DEG C are extended 5min, after PCR finishes, get 8 μ l products and do agarose gel electrophoresis analysis.Result as shown in Figure 3.
Fig. 3 carries out multiple PCR primer specific amplification result figure to difference test sample.M is standard molecular weight sample, and 1,2 is that two viruses infect sample simultaneously, and 3,4 infect separately sample for TGEV.
If what the double PCR method of the present invention that illustrates Fig. 3 detected is the sample of single virus infection, can only increase one, if for the detected sample that has infected Geminivirus, must amplify two bar segment.The PCR detection method of its more existing report has improved detection efficiency, and high specificity.
Embodiment 2 verifies the specificity experiment of multiplex PCR.
1RNA extracts
Method is with comparative example 1.
2cDNA is synthetic
Method is with comparative example 1, and to extract, pig breathes and RNA the reverse transcription of breeding syndrome virus (PRRSV), Pestivirus suis (CSFV) and porcine rotavirus (PRAV) obtain cDNA, carries out multiple RT-PCR react with the cDNA template of PEDV and TGEV.
The detection of 3 multiplex PCRs
Taking the synthetic cDNA of step 2 as template, 2 pairs of primers that sequence are respectively to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, SEQ ID NO.4 add amplification system simultaneously, set up double pcr amplification reaction system.Double PCR reaction system is as follows: cDNA template 1 μ l, and 2 couples of each 0.5 μ l of upstream and downstream primer, PCRMix12.5 μ l, ultrapure water is supplemented to 50 μ l.PCR reaction conditions: 95 DEG C of denaturation 5min, enter 95 DEG C of 60s, the circulation of 52 DEG C of 1min and 72 DEG C of 1min, circulates 35 times, and last 72 DEG C are extended 5min, after PCR finishes, get 8 μ l products and do agarose gel electrophoresis analysis.
Fig. 4 is multiple PCR primer specific amplification result figure.M is standard molecular weight sample, and 1,2 for doubtful two TGEV and PEDV infect sample simultaneously, and 3 is PRRSV, and 4 is CSFV, 5PRAV.Presentation of results multiplex PCR specificity is very strong.
Claims (3)
1. a double PCR method that detects transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus, is characterized in that, concrete steps are as follows:
(1) from infecting the positive intestinal tube tissue extraction RNA of TGEV, PEDV, reverse transcription becomes cDNA, further taking cDNA as masterplate, by add PCR reaction system for 2 pairs of primers of TGEV and PEDV design respectively simultaneously, carries out pcr amplification;
(2) after PCR finishes, PCR product is done to agarose gel electrophoresis analysis, obtain 385bp specific gene fragment and represent infected pigs's Transmissible gastroenteritis virus, obtain 628bp specific gene fragment and represent infected pigs's epidemic virus; Wherein:
The sequence of described 2 pairs of primers is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, SEQ ID NO.4.
2. double PCR method according to claim 1, is characterized in that: in step (1), PCR reaction system is as follows: cDNA template 1 μ l, and 2 couples of each 0.5 μ l of upstream and downstream primer, PCR Mix12.5 μ l, ultrapure water is supplemented to 50 μ l.
3. double PCR method according to claim 1, is characterized in that: in step (1), pcr amplification reaction condition is as follows: 95 DEG C of denaturation 5min, enter 95 DEG C of 60s, and the circulation of 52 DEG C of 1min and 72 DEG C of 1min, circulates 35 times, and last 72 DEG C are extended 5min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328223A (en) * | 2014-11-26 | 2015-02-04 | 哈药集团生物疫苗有限公司 | RT-PCR primer group and RT-PCR kit for simultaneously detecting four porcine RNA viruses |
| CN104611465A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus |
| CN105154583A (en) * | 2015-07-14 | 2015-12-16 | 天津瑞普生物技术股份有限公司 | Double-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously identifying TGEV (transmissible gastroenteritis viruses) and PEDV (porcine epidemic diarrhea viruses) |
| CN105543416A (en) * | 2016-01-28 | 2016-05-04 | 北京佰鸥创投生物科技有限公司 | Double microdroplet digital PCR (Polymerase Chain Reaction) absolute quantitative detection kit for porcine epidemic diarrhea virus and transmissible gastroenteritis virus |
| CN108531658A (en) * | 2018-05-09 | 2018-09-14 | 龙岩学院 | A kind of detection method of PEDV and TGEV primer sequences and duplex RT-PCR |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328223A (en) * | 2014-11-26 | 2015-02-04 | 哈药集团生物疫苗有限公司 | RT-PCR primer group and RT-PCR kit for simultaneously detecting four porcine RNA viruses |
| CN104611465A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus |
| CN105154583A (en) * | 2015-07-14 | 2015-12-16 | 天津瑞普生物技术股份有限公司 | Double-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously identifying TGEV (transmissible gastroenteritis viruses) and PEDV (porcine epidemic diarrhea viruses) |
| CN105543416A (en) * | 2016-01-28 | 2016-05-04 | 北京佰鸥创投生物科技有限公司 | Double microdroplet digital PCR (Polymerase Chain Reaction) absolute quantitative detection kit for porcine epidemic diarrhea virus and transmissible gastroenteritis virus |
| CN108531658A (en) * | 2018-05-09 | 2018-09-14 | 龙岩学院 | A kind of detection method of PEDV and TGEV primer sequences and duplex RT-PCR |
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