CN103952496B - A kind of duplex PCR method detecting transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus - Google Patents
A kind of duplex PCR method detecting transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention belongs to biological technical field, be specially a kind of duplex PCR method detecting transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus.The present invention designs two pairs of primers of the specific sequence of increase respectively TGEV, PEDV, from the positive intestinal tube tissue extraction RNA infecting TGEV, PEDV, reverse transcription becomes cDNA, is masterplate further with cDNA, establishes the multi-PCR detection method of directly one-time detection two kinds of diarrhea viruses from sample.Beneficial effect of the present invention is: the PCR detection method of its more existing report improves detection efficiency, and high 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 duplex PCR method detecting transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus in porcine tissue.
Background technology
Transmissible gastroenteritis of swine (transmissiblegastroenteritis, TGE) be by the Transmissible gastroenteritis virus (transmissiblegastroenteritisvirus of coronaviridae, a kind of contagious disease of the pig TGEV) caused, in 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 to lose to pig industry.Its clinical with vomiting, severe diarrhea and dehydration property enteritis for principal character.The equal susceptible of pig of different ages and kind, especially to the piglet within 2 week age, its lethality rate is up to 100%.TGE virus belongs to coronaviridae coronavirus genus.
Porcine epizootic diarrhea (Porcineepidemicdiarrhea, PED) be by the Porcine epidemic diarrhea virus (porcineepidemicdiarrheavirus of coronaviridae coronavirus genus, PEDV) pig caused acute, high degree in contact infectious intestinal disease, China just had the report of PED successively from 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 occurs that serious grice diarrhoea is popular, cause the death of a large amount of sucking piglets, also huge financial loss is caused to pig industry.Its clinical with watery diarrhea, vomiting, dehydration for principal character, 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, the heavy losses such as viral diarrhea causes that the pig farm price of deed reduces, 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 usually need by laboratory diagnostic technique, as virus purification, immunofluorescent test, round pcr etc.Traditional diagnostic techniques such as virus purification, immunofluorescent test etc. are wasted time and energy, and are unsuitable for clinical quick diagnosis, are also unsuitable for large-scale epidemiology survey.And round pcr has high specificity, susceptibility is high, can carry out the features such as vivo diagnostic, has comparatively outstanding advantage in laboratory diagnosis.
Multiplex PCR (multiplexPCR) is improved on the basis of regular-PCR, adopts the different zones of multipair Auele Specific Primer to multiple DNA profiling or same template to increase the round pcr of multiple object fragment.It is by adding that in same PCR reaction tubes the Auele Specific Primer of multiple pathogenic microorganisms carries out pcr amplification simultaneously, and can detect multiple pathogens simultaneously or identify is that type pathogenic infection.
Because multiplex PCR increases multiple goal gene simultaneously, there is the advantage of saving time, reducing costs, raising the efficiency, particularly save precious laboratory sample, so once proposition, namely the favor of numerous investigator is obtained, and development rapidly, in the every field of life science, multiplex PCR has become a maturation and important research means.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide the multi-PCR detection method of a kind of direct one-time detection transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus from sample, the PCR detection method of its more existing report improves detection efficiency.
For two kinds of virus polyinfections often in actual production, very indistinguishable situation, the present invention utilizes information biology means, from the various factors analyzing gene fragment structure affecting PCR detection sensitivity, filter out the easiest gene segment gone out by pcr amplification and detect target spot as PCR, analyze from primer mass parameter aspect, the primer of these target gene of design amplification; Two couple of design is utilized to increase respectively to primer the specific sequence of TGEV, PEDV further, with TGEV and (or) PEDV suspected infection sample extraction RNA, send out further and be transcribed into cDNA, be amplification template with cDNA, establish duplex PCR system transmissible gastroenteritis of swine, porcine epizootic diarrhea being infected to differential diagnosis.Its concrete technical scheme is described below.
The invention provides a kind of duplex PCR method of Transmissible gastroenteritis virus and Porcine epidemic diarrhea virus, concrete steps comprise:
(1) from the positive intestinal tube tissue extraction RNA infecting TGEV, PEDV, reverse transcription becomes cDNA, is masterplate further with cDNA, 2 pairs of primers respectively for TGEV and PEDV is added PCR reaction system simultaneously, carries out pcr amplification;
(2) after PCR terminates, agarose gel electrophoresis analysis is done to PCR primer, 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 SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3 and SEQIDNO.4:
Upstream primer TGEV:5 ' CAACCCCGATGGAGCACT3 ';
Downstream primer TGEV:5 ' CCGAACATTACATATCTGGACACT3 ';
Upstream primer PEDV:5 ' TGAGGGTGACAAGTTCGTAGG3 ';
Downstream primer PEDV:5 ' CGCCAAGATATTAAAAGATTCACC3 ';
In above-mentioned steps (1), PCR reaction system is as follows: cDNA template 1 μ l, 2 couples of each 0.5 μ l, PCRMix12.5 μ l of upstream and downstream primer, and ultrapure water is supplemented to 50 μ l; Pcr amplification reaction condition is as follows: 95 DEG C of denaturation 5min, enters 95 DEG C of 60s, the circulation of 52 DEG C of 1min and 72 DEG C 1min, circulates 35 times, and last 72 DEG C extend 5min.
Compared with prior art, the gene fragment that the present invention utilizes oneself to screen detects target spot as ideal, explore in annealing temperature, primer and other reaction system parameters etc., filter out multiplex PCR detection system that is highly sensitive, high specificity, set up the multi-PCR detection method of directly one-time detection two kinds of diarrhea viruses from sample, experiment proves, the PCR detection method of more existing report, multi-PCR detection method of the present invention is reproducible, high specificity, detection efficiency are high.
Accompanying drawing explanation
Fig. 1 is the single pcr amplification result figure that comparative example 1 tests sample.M is standard molecular weight sample, and 1,2 is two suspected infection PEDV viral sample, consistent with the object fragment (628bp) of prediction; 3,4 is negative control.
Fig. 2 is the single pcr amplification result figure that comparative example 1 tests sample.M is standard molecular weight sample, and P4 is TGEV virus infection sample, consistent with the object fragment (385bp) of prediction; P3 is negative control.
Fig. 3 is that embodiment 1 difference test sample carries out multiple PCR primer specific amplification result figure.M is standard molecular weight sample, and 1,2 is that two viruses infect sample, 3 simultaneously, and 4 infect sample separately for TGEV.
Fig. 4 is multiple PCR primer specific amplification result figure in embodiment 2.M is standard molecular weight sample, and 1,2 infect sample for doubtful two TGEV and PEDV simultaneously, and 3 is PRRSV, and 4 is CSFV, 5PRAV.
Embodiment
The present embodiment is implemented under premised on technical solution of the present invention, gives 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, usually conveniently condition, or according to the condition that reagent manufacturer advises.
The present invention utilizes a kind of information biology means, first filters out the easiest virus (TGEV and the PEDV) gene segment gone out by pcr amplification and detects target spot as PCR, and then the primer of these target gene of design amplification, and 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, in Nucleotide mono-hurdle of homepage, first input two goal gene that will look for respectively, the complete genome sequence had from database afterwards downloads.
Porcineepidemicdiarrheavirus，completegenome
27，953bpIinearRNA
Accession：KC189944.1GI：509265008
GenBankFASTAGranhics
TGEVvirulentPurdue，completegenome
28，544bpIinearRNA
Accession：DQ811789.2GI:331265425
GenBankFASTA
GranhicsRelatedSequences
2) design of primers of virus (TGEV and PEDV) tested gene target
The document of the complete sequence of above-mentioned these two virogenes found after order-checking is opened by DNAstar software, according to DNAstar working instructions, after sequence is read, according to setting, software requires that (dimer hairpin structure is no more than 1, annealing temperature is between 48 to 55 degree) screen, again the primer scheme that gene order is provided by software be optimized and mate, thus designing the primer of virus (TGEV and PEDV) tested gene target; The sequence of 2 pairs of primers is respectively as shown in SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3 and SEQIDNO.4:
Upstream primer TGEV:5 ' CAACCCCGATGGAGCACT3 ';
Downstream primer TGEV:5 ' CCGAACATTACATATCTGGACACT3 ';
Upstream primer PEDV:5 ' TGAGGGTGACAAGTTCGTAGG3 ';
Downstream primer PEDV:5 ' CGCCAAGATATTAAAAGATTCACC3 ';
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 Trizol used method and extracts.Method reference reagent box specification sheets.The first step take certain pig farm suspected infection TGEV or (with) PEDV pig intestinal tissue 20g, grind, add TrizolReagent200 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, 4 DEG C, the centrifugal 15min of 12000r/min, draws upper strata aqueous phase, in another centrifuge tube.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, abandon supernatant, RNA is sunken at the bottom of pipe.4th step, adds 1mL75% ethanol, 1mlTrizol, gentle vibration centrifuge tube, and suspend precipitation, washing RNA precipitation.5th step, 4 DEG C, the centrifugal 5min of 8000r/min.Abandon supernatant, room temperature is dried or vacuum-drying 5-10min, makes abundant drying as far as possible, generally can see that some transparent materials are at the bottom of pipe facing to light.Add 16 μ LDEPC water dissolution.DEPC water can the various protein of deactivation, is the potent inhibitor of RNA enzyme, can be used for RNA resolution of precipitate.
2cDNA synthesizes
Take RNA as template, synthesis cDNA chain.The first step, adds 8 μ lRNA templates and 2 μ lPrimeScript in 1.5mL centrifuge tube
tM(ThermoScript II) RTMasterMix (PerfectRealTime).Reaction conditions 37 DEG C of 15min, 85 DEG C of 5s, 4 DEG C of 5min, reaction terminates rear ice bath and preserves.Obtain cDNA.
3 single pathogen gene pcr amplifications
With the cDNA of the 2-in-1 one-tenth of step for template, be SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3 respectively with sequence, 2 pairs of primers of SEQIDNO.4 do single amplification to primer.Amplification system is each 0.5 μ l of upstream and downstream primer, ultrapure water 10.5 μ l, and PCRMix(Shanghai Sheng Gong biotechnology company limited purchases) 12.5 μ l, cDNA template 1 μ l, have the mixture of 25 μ l in single system.95 DEG C of denaturation 5min, then continue 95 DEG C of 60s, 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 the circulation of 72 DEG C of 60s, circulate 30 times altogether, last 72 DEG C extend 5min.Preserve PCR primer for 4 DEG C.After PCR terminates, 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 synthesizes
Method is with comparative example 1.
The foundation of 3 multiplex PCRs
With suspected infection TGEV or (with) the sample extraction RNA of PEDV, transcribe and obtain cDNA, with this cDNA for template, sequence is respectively SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3,2 pairs of primers of SEQIDNO.4 add amplification system simultaneously, set up duplex PCR amplification reaction system.Duplex PCR reaction system is as follows: cDNA template 1 μ l, and 2 couples of each 0.5 μ l, PCRMix12.5 μ l of upstream and downstream primer, ultrapure water is supplemented to 50 μ l.PCR reaction conditions: 95 DEG C of denaturation 5min, enters 95 DEG C of 60s, the circulation of 52 DEG C of 1min and 72 DEG C 1min, circulates 35 times, after last 72 DEG C of extensions 5min, PCR terminate, gets 8 μ l products and does 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, 3 simultaneously, and 4 infect sample separately for TGEV.
If the sample of what the duplex PCR method of the present invention that illustrates Fig. 3 detected is single virus infection, then can only increase one, if for the detected sample infecting Geminivirus, must amplify two bar segment.The PCR detection method of its more existing report improves detection efficiency, and high specificity.
Embodiment 2 verifies the specificity experiments of multiplex PCR.
1RNA extracts
Method is with comparative example 1.
2cDNA synthesizes
Method, with comparative example 1, to be breathed with the RNA of reproductive syndrome virus (PRRSV), Pestivirus suis (CSFV) and porcine rotavirus (PRAV) and reverse transcription obtains cDNA to extract pig, is carried out multiple RT-PCR reaction with the cDNA template of PEDV and TGEV.
The detection of 3 multiplex PCRs
With the cDNA of the 2-in-1 one-tenth of step for template, sequence is respectively SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3,2 pairs of primers of SEQIDNO.4 add amplification system simultaneously, set up duplex PCR amplification reaction system.Duplex PCR reaction system is as follows: cDNA template 1 μ l, and 2 couples of each 0.5 μ l, PCRMix12.5 μ l of upstream and downstream primer, ultrapure water is supplemented to 50 μ l.PCR reaction conditions: 95 DEG C of denaturation 5min, enters 95 DEG C of 60s, the circulation of 52 DEG C of 1min and 72 DEG C 1min, circulates 35 times, after last 72 DEG C of extensions 5min, PCR terminate, gets 8 μ l products and does agarose gel electrophoresis analysis.
Fig. 4 is multiple PCR primer specific amplification result figure.M is standard molecular weight sample, and 1,2 infect sample for doubtful two TGEV and PEDV simultaneously, and 3 is PRRSV, and 4 is CSFV, 5PRAV.Result illustrates that multiplex PCR specificity is very strong.
Claims (3)
1. detect a duplex PCR primer sets for transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus, it is characterized in that, the sequence of primer sets is respectively as shown in SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3, SEQIDNO.4; By as follows for the concrete steps that two pairs of primers are used for detecting transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus:
(1) from the positive intestinal tube tissue extraction RNA infecting TGEV, PEDV, reverse transcription becomes cDNA, is masterplate further with cDNA, by adding PCR reaction system for the primer sets of TGEV and PEDV design respectively simultaneously, carries out pcr amplification;
(2) after PCR terminates, agarose gel electrophoresis analysis is done to PCR primer, 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.
2. duplex PCR primer sets according to claim 1, is characterized in that: in step (1), PCR reaction system is as follows: cDNA template 1 μ l, 2 couples of each 0.5 μ l, PCRMix12.5 μ l of upstream and downstream primer, ultrapure water is supplemented to 50 μ l.
3. duplex PCR primer sets according to claim 1, it 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, the circulation of 52 DEG C of 1min and 72 DEG C 1min, circulate 35 times, last 72 DEG C extend 5min.
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| 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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