CN107419034B - Five-porcine diarrhea virus multiple PCR rapid diagnosis kit and application thereof - Google Patents
Five-porcine diarrhea virus multiple PCR rapid diagnosis kit and application thereof Download PDFInfo
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Abstract
The invention discloses a five-porcine diarrhea virus multiplex PCR rapid diagnosis kit, which comprises 5 pairs of PCR primers, and also comprises PCR reaction liquid, a PCR standard substance, a positive reference substance and a negative reference substance. The invention also discloses application of the kit in simultaneous specific detection of five porcine diarrhea viruses, wherein the five porcine diarrhea viruses are TGEV, GAR, GCR, PEDV and PCV 2.
Description
Technical Field
The invention belongs to the field of virus nucleic acid detection, and particularly relates to a five-pig diarrhea virus multiplex PCR (polymerase chain reaction) rapid diagnosis kit which can simultaneously detect five pig viruses including Porcine Epidemic Diarrhea Virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine rotavirus A (GAR), Porcine rotavirus C (GCR) and Porcine circovirus 2 (PCV2) in the same reaction tube, and is suitable for rapid detection of the five pig viruses in clinical and scientific researches.
Background
In recent years, with the development of the pig industry towards scale and intensification, the situation that the swinery has multi-pathogen mixed infection and secondary infection is more and more common. The diseases infected with Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus A (GAR), porcine rotavirus C (GCR) and porcine circovirus 2 (PCV2) present similar diarrhea symptoms clinically, and are often mixed infections, and the occurrence of the diseases complicates and makes difficult the diagnosis of other diseases such as respiratory and reproductive disorders, has high mortality rate, and causes great loss to the pig industry.
Porcine Epidemic Diarrheal (PED) is an enteric infectious disease of pigs caused by Porcine Epidemic Diarrheal Virus (PEDV) which is characterized primarily by vomiting, diarrhea and dehydration. The disease mainly occurs to piglets within 7 days of age, has short course of disease, rapid spread and extremely high piglet mortality rate which can reach 100 percent. In 1978, Belgium and the UK reported the epidemic of PEDV for the first time, and in 1984, China confirmed the existence of the disease. Big outbreaks of PEDV are reported in 2010-2012 in different areas of China, so that a large number of suckling piglets die, and the healthy development of the pig industry in China is seriously influenced.
The genome of the transmissible gastroenteritis virus (TGEV) belongs to the family of coronaviridae and the genus coronavirus is single-strand positive-strand non-segmented RNA with the total length of 28.5 kb. Causing damage to the small intestine, resulting in dyspepsia and malabsorption, eventually leading to diarrhea and dehydration, causing death of the suckling piglets (Tuboyy et al, 2000; south China and Mongolia, 2010). The virus infects high rates, resulting in high mortality of piglets (Wang Li et al, 2015).
Rotavirus (RV) belongs to the family reoviridae, and is a double-stranded RNA with 11 segments, with a single genome, without an envelope (Estes and Cohen, 1989). RV is an important factor causing diarrhea in humans as well as other animals, and rotavirus is currently classified into 8 species by the VP6 gene, of which A, B, C, E and H (GAR, GBR, GCR, GER and GHR) have been detected in swine (Matthijnssens et al, 2012; Saif et al, 1980; Wakuda et al, 2011). GAR was first reported in 1975 to have been consistently recognized as a major cause of porcine diarrhea (Amimo et al, 2013 a; Ciarlet et al, 2002; Ciarlet et al, 1995; Miyazaki et al, 2011, 2012). GCR was first detected in 1979 and was difficult to culture (Amimo et al, 2013 b; Marthaler et al, 2013; Saif et al, 1988; Saif et al, 1996; Tsunemitsu et al, 1991). GCR can cause severe gastrointestinal infections and is prone to sporadic transmission or outbreaks (Bohl et al, 1982; Collins et al, 2008; Saif et al, 1980). GAR is generally considered to be the leading cause of pig diarrhea, while GBR and GCR have less significant effects. A study of Douglas marthaler and the like on RT-PCR detection of 7508 porcine diarrhea samples finds that the GAR positive rate is 62%, the GCR infection rate is also very high (53%), GCR mainly infects piglets for 1-20 days, and GAR mainly infects piglets for more than 21 days. It is believed that GAR, GCR (Marthalter et al, 2014) should be detected simultaneously.
Porcine circovirus type 2 (PCV2) genus circovirus of the circovirus family, a recently newly reported porcine virus, is a minimal single-stranded circular DNA virus capable of autonomous replication in mammalian cells, comprising 4 Open Reading Frames (ORFs): ORF1-4(Murphy, 1995). One of the symptoms of porcine circovirus-associated disease (PCVAD) is the induction of enteritis. PCV2 can cause enteritis either by infection alone or in combination (Kim et al, 2004; Zhang et al, 2013). PCV2 has strong infectivity on pigs, can infect pigs at all ages, and invades the immune system of the pigs to generate immunosuppression and secondary infection of other pathogenic microorganisms.
The conventional etiology and serology detection is difficult to distinguish and identify the diseases simultaneously, and has the defects of complex operation, time and labor waste, low sensitivity and the like, and particularly, when a plurality of pathogenic infections are infected, the early detection is difficult to effectively carry out, and misjudgment is easy to generate. The multiplex PCR technology can simultaneously detect a plurality of pathogens at one time, has the advantages of high specificity and sensitivity, short detection time, low detection cost and the like, and is an ideal detection method. Therefore, the multiplex PCR rapid diagnosis kit for simultaneously and specifically detecting the infection of various porcine diarrhea viruses is developed, is beneficial to improving the detection level of the porcine diarrhea viruses, and has important application prospects in the aspects of disease diagnosis, prevention and treatment, food safety and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a five-porcine diarrhea virus multiplex PCR rapid diagnosis kit and application thereof.
In order to solve the technical problems, the invention provides a five-porcine diarrhea virus multiplex PCR rapid diagnosis kit:
comprises the following 5 pairs of PCR primers (five virus-specific primers):
TGEV-F：GTGGTGTTAGGTGATTATTTTCC，
TGEV-R：TATGGTTTAACCTGCACTCACTA；
GAR-F：TATGCAATACCAGTAGGACCAG，
GAR-R：GCTCTACGTAGCGAGTATGAAATC；
PEDV-F：AACACGGCGACTACTCAGC，
PEDV-R：GCCTTCTTTAGCAACCCAG；
GCR-F：TGTTGCATCCGTGAAGAGAATGGT；
GCR-R：GCATTAGCCCCTACGCAAGC；；
PCV2-F：AAGAAGCGGACCCCAAC，
PCV2-R：AGGTGGCCCCACAATGA。
the five porcine diarrhea virus multiplex PCR rapid diagnosis kit is improved as follows:
the kit comprises: a) a multiplex PCR reaction solution, b) a primer (a virus-specific primer), c) a positive control, d) a negative control and e) a PCR standard;
a) the PCR reaction solution (i.e., multiplex PCR reaction mixture) comprises a PCR reaction buffer and a thermostable DNA polymerase; the PCR reaction buffer solution comprises a buffer solution, a mixture of magnesium chloride and deoxyribonucleotide triphosphate;
b) the primers are five virus specific primers;
c) and the positive control substance is: a positive plasmid cocktail containing five viruses TGEV, GAR, GCR, PEDV and PCV2 (each virus at a concentration of about 300 ng/. mu.l);
d) and the negative reference substance is: disinfection of diethyl pyrocarbonate treated Water (DEPC-H)2O)；
e) The PCR standard substance consists of the following 5 standard substances: PEDV standard, TGEV standard, GAR standard, GCR standard, PCV2 standard.
Remarks explanation:
the sequences of the 5 virus standard products are shown in SEQ ID NO. 11-SEQ ID NO. 15.
The five porcine virus multiplex PCR rapid diagnosis kit is further improved as follows:
the upstream and downstream primers of the specific primers of the five viruses are packaged in the same tube.
The diagnostic kit can be used for detecting Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus A (GAR), porcine rotavirus C (GCR) and porcine circovirus 2 (PCV 2).
The rapid diagnostic kit provided by the invention is stored at-20 ℃ in the dark, and repeated freeze thawing is reduced as much as possible.
TABLE 1 multiplex PCR primers designed in the present invention
The use method of the kit can specifically comprise the following steps:
setting a negative control and a positive control according to specific use conditions in each detection;
diluting the standard substance with sterile double distilled water to 1.0 × 101～1.0×108Copies/μl。
RNA/DNA virus sample extraction: viral nucleic acids were extracted from cell lines, fresh or frozen samples using the Viral RNA/DNA Extraction Kit MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0(TAKARA) according to the instructions for use of the product.
Reverse transcription reaction 1. mu.l of the total RNA/DNA template prepared in the previous step was sequentially added to a 0.2ml RNase-free PCR tube, and RT-PCR reaction was performed using TaKaRa RNA PCR Kit (AMV) Ver.3.0(Code No. RR019A) in which 10 × RT PCR Buffer 1. mu.l, 25mM MgCl22μl，10mM dNTP Mixture 1μl，40U/μl RNaseInhibitor 0.25μl，5U/μl AMV RTase XL 0.5μl，Random 9 mers 0.5μl，RNase Free dH2And O till the total volume is 10 mu l, and obtaining reaction liquid which is the cDNA/DNA template after the reverse transcription reaction is finished.
Detection of nucleic acids: taking 1. mu.l of prepared cDNA/DNA as a template to carry out multiple fluorescence PCR reaction, wherein 19. mu.l of multiple PCR reaction solution, 1. mu.l of primer mixture and ddH2O4 mu l; the reaction procedure is firstly denaturation at 95 ℃ for 3 min; the reaction conditions of 40 cycles are denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30 s; finally 5min at 72 ℃. The PCR product was detected by 2% agarose gel electrophoresis and analyzed after imaging by a gel imager.
And (3) reporting a result: whether the five porcine diarrhea viruses exist is identified according to the existence of target fragments of TGEV, GAR, GCR, PEDV and PCV2 genes.
The invention has the advantages that: by using a multiplex PCR technology, specific primers of five viruses of TGEV, GAR, GCR, PEDV and PCV2 are designed, and the developed five porcine diarrhea virus multiplex PCR rapid diagnosis kit can simultaneously detect single or mixed infection viruses of TGEV, GAR, GCR, PEDV and PCV2 from a sample through a PCR reaction, has high specificity, and is simpler, more time-saving and lower in cost than the traditional common PCR method. The kit provided by the invention can distinguish porcine diarrhea virus infection and provide a quick and simple tool for detecting the porcine diarrhea virus infection, thereby realizing clinical early diagnosis and timely formulating a treatment scheme and reducing the mortality rate and economic loss of swinery.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the specific detection of primers corresponding to five porcine diarrhea viruses. The lanes are from left to right: m is DL2000 DNAmarker; 1: GCR; GAR; 3, TGEV; 4, PEDV; 5: PCV 2; GCR, GAR, TGEV, PEDV and PCV 2; GCR and TGEV; GAR and PEDV; TGEV and PCV 2; GCR and PCV 2; GCR, GAR, and TGEV; 12: TGEV, PEDV and PCV 2; GCR, TGEV and PCV 2; GCR, TGEV, PEDV and PCV 2; 15: GAR, TGEV, PEDV, and PCV 2; 16: negative control; non-target template 17.
FIG. 2 shows the sensitivity detection of the present invention for five porcine diarrhea viruses, lanes 1-9 are sequentially from left to right 1:5 × 105；2:5×104；3:5×103；4:5×102；5:5×101；6:5×100；7:5×10-1；8:5×10-2copies/. mu.L standard mix; 9 negative control.
Detailed Description
The invention will be further described with reference to the following figures and examples, but the scope of the invention is not limited thereto.
Example 1 kit Structure
A five-porcine virus multiplex real-time fluorescent quantitative PCR rapid diagnosis kit comprises multiplex PCR reaction mixed liquor, virus primers, TGEV virus standard substance, GAR virus standard substance, GCR virus standard substance, PEDV virus, PCV2 virus standard substance, positive control substance, negative control substance and a kit body.
The box body is provided with corresponding container holes for respectively placing a quantitative PCR reaction liquid tube, five virus primer tubes, a TGEV virus standard tube, a GAR virus standard tube, a GCR virus standard tube, a PEDV virus standard tube, a PCV2 virus standard tube, a positive control tube and a negative control tube.
Wherein the mixed solution of the multiple PCR reaction comprises a PCR reaction buffer solution (containing a mixture of magnesium chloride and deoxyribonucleotide triphosphates, etc.) and heat-resistant DNA polymerase; the virus primers are five virus primers, and the upstream primers and the downstream primers are packaged in the same tube; the positive control is positive plasmid mixture of five viruses of TGEV, GAR, GCR, PEDV and PCV2 (each virus concentration is about 300 ng/. mu.l); the negative control is sterilized diethyl pyrocarbonate treated water (DEPC-H)2O)。
Example 2 specificity test for detecting five viruses
The first step is as follows: primer synthesis
5 types of virus primer sequences designed by the invention (see table 1) are synthesized into primers by technical service company of Shanghai Biotech company in China, and the synthesis amount is 3OD per primer.
The second step is that: multiplex PCR system specific detection
According to a multiple PCR reaction system, taking 17 parts of the same PCR reaction premix (namely, 19 mu L of multiple PCR reaction mixture and 1 mu L of 5 virus primer mixture), and respectively adding 1 mu L of positive sample cDNA/DNA template 1: GCR; GAR; 3, TGEV; 4, PEDV; 5: PCV 2; GCR, GAR, TGEV, PEDV and PCV 2; GCR and TGEV; GAR and PEDV; TGEV and PCV 2; GCR and PCV 2; GCR, GAR, and TGEV; 12: TGEV, PEDV and PCV 2; GCR, TGEV and PCV 2; GCR, TGEV, PEDV and PCV 2; 15: GAR, TGEV, PEDV, and PCV 2; 16: negative control of deionized water; 17 non-target template, plus ddH2O to a total volume of 25. mu.L. The reaction is carried out in a Bio-Rad S1000 PCR amplification instrument, and the reaction parameters are as follows: first at 95 deg.CSex 3 min; the reaction conditions of 40 cycles are denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30 s; finally 5min at 72 ℃. Taking 5 mu L of PCR product, mixing with 1 mu L of Loading Buffer, spotting in a 2% agarose gel electrophoresis plate hole, carrying out electrophoresis at 120V for 30min, and taking a picture under an ultraviolet fluorescence phase former for judgment, wherein the result is shown in figure 1, the sizes of the corresponding PCR products are respectively 126bp, 242bp, 319bp, 394bp and 508bp which are respectively corresponding to GCR, GAR, TGEV, PEDV and PCV2 in the table 1, and the specificity of the porcine diarrhea virus multiplex PCR reaction system constructed in the invention is better.
Example 3 test for the sensitivity of the invention to five viruses
The first step is as follows: primer synthesis
The same as the first step in example 2.
The second step is that: sensitivity detection
The multiplex PCR sensitivity detection system comprises 19 μ L of PCR reaction solution (multiplex PCR reaction mixture), 10 μ M of TGEV, GAR, GCR, PEDV and PCV2 upstream and downstream primer mixture 1 μ L, 1 μ L of TGEV, GAR, GCR, PEDV and PCV2 plasmid standard substance (5.0 × 10) diluted by 10 times in series8～5.0×101copies/. mu.L) template, plus ddH2O till the total volume of the reaction system is 25 mu L; the reaction is carried out in a Bio-Rad S1000 PCR amplification instrument, and the reaction parameter is denaturation at 95 ℃ for 3 min; 40 cycles of denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30 s; finally 5min at 72 ℃. mu.L of PCR product was mixed with 1. mu.L of LoadingBuffer and detected by 2% agarose electrophoresis.
A conventional single PCR sensitivity detection reaction system comprises 2.5 mu L of 10 × PCR buffer and 25mM MgCl22.5 μ L, 2.5 μ M dNTP 2 μ L, Taq DNA Polymerase (Shanghai Producer) 1.25U, each diarrhea virus primer described in Table 1, final concentration of upstream and downstream primers was 0.2 μ M, 10-fold serial dilution of each viral plasmid standard (5.0 × 10)8～5.0×101copies/. mu.L) template, plus ddH2O till the total volume of the reaction system is 25 mu L; the reaction is carried out in a Bio-Rad S1000 PCR amplification instrument, and the amplification reaction parameters are as follows: 5min at 95 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃, 40 cycles, and 10min at 72 ℃. mu.L of the amplification product was subjected to 1% agarose electrophoresis.
The test results show that the minimum detection amount of GCR, GAR, TGEV, PEDV and PCV2 in the multiplex PCR reaction is 50copies, and the results are shown in FIG. 2, while the minimum detection amount of GCR in the single PCR reaction is 5 × 102copies, GAR 5 × 103copies, TGEV 5 × 102copies, PEDV 5 × 102copies, PCV2 is 5 × 102copies。
Therefore, the sensitivity of the multiplex PCR detection of the diarrhea virus is 10-100 times higher than that of the single PCR, and is obviously higher than that of the multiplex PCR detection of four diarrhea viruses (GAR is 1.26 × 10) reported by Zhao et al (2013, Journal of Virological Methods)4copies, TGEV 1.74 × 104copies, PEDV 2.1 × 103copies, PCV2 was 2.17 × 103copies) approximately 250-40 times higher.
Example 4 clinical test of five porcine diarrhea viruses of the invention
The first step is as follows: primer synthesis
The same as the first step in example 2.
The second step is that: sample collection
The total amount of the serum samples is 78 parts, wherein 53 parts of excrement and 25 parts of tissue disease materials are collected in Zhejiang.
The third step: total DNA/RNA miniprep extraction
Viral nucleic acids were extracted from samples collected in the second step, fresh or frozen, using the Viral RNA/DNA extraction Kit MiniBEST Viral RNA/DNAextraction Kit Ver.4.0(TAKARA) according to the instructions for use of the product.
The fourth step: reverse transcription to synthesize cDNA/DNA:
reverse transcription reaction: sequentially adding 2 mu l of the total RNA/DNA template prepared in the previous Step into a 0.2ml PCR tube without RNase, carrying out RT-PCR reaction according to the operation instruction of TaKaRa One Step RNA PCR Kit (AMV) (Code No. RR024A), and obtaining reaction liquid, namely the cDNA/DNA template after the reverse transcription reaction is finished.
The fifth step: general PCR detection and multiplex PCR
1. Ordinary PCR detection, PCR reaction system (total reaction volume 25. mu.l), 10 × PCR buffer 2.5. mu.l, 25mM MgCl22.5μl，2.5 μ M dNTP 2 μ l, Taq DNA polymerase 1.25U, final concentration of 0.2 μ M of five viral primers synthesized in the first step, cDNA/DNA (about 100ng) synthesized in the fourth step as template, and ddH2O to a total volume of 25. mu.L. The reaction is carried out in a Bio-Rad S1000 PCR amplification instrument, and the reaction parameters are as follows: 3min at 95 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃, 40 cycles, and 10min at 72 ℃. mu.L of the amplification product was subjected to 1% agarose electrophoresis.
2. The multiplex PCR detection system is as follows: mu.L of PCR reaction mixture (i.e., multiplex PCR reaction mixture), 10. mu.M of TGEV, GAR, GCR, PEDV and PCV2 upstream and downstream primer mixture 1. mu.L, 1. mu.L of cDNA/DNA template synthesized in the fourth step, plus ddH2O till the total volume of the reaction system is 25 mu L; the reaction parameters are 95 ℃ denaturation for 3 min; 40 cycles of denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30 s; finally 5min at 72 ℃.5 mu L of PCR product is mixed with 1 mu L of Loading Buffer and detected by 2% agarose electrophoresis.
And a sixth step: the result of the detection
The detection results of the common PCR method and the detection results of the invention are as follows: 38 parts of PEDV is detected by common PCR, and 46 parts of PEDV is detected by the method; PCV 231 parts are detected by common PCR, and positive PCV 39 parts are detected by the method; 7 portions of GCR are detected by common PCR, and 7 portions of positive are detected by the invention; common PCR detects 3 parts of TGEV, and the invention detects 4 parts of positive; 1 part of GAR is detected by common PCR, and 3 parts of positive GAR is detected by the method; wherein 12 parts of mixed infection of PEDV and PCV2 is detected by the invention, and 9 parts of mixed infection of PEDV and PCV2 is detected by ordinary PCR; 2 parts of mixed infection of PEDV and GCR is detected, and 1 part of mixed infection of PEDV and PCV2 is detected by common PCR; the invention detects 1 part of mixed infection of TGEV and PCV2, and common PCR detects 0 part of mixed infection of TGEV and PCV 2.
The detection effect of the invention is obviously better than that of single PCR, the invention can reduce the occurrence of false negative, simultaneously can detect mixed infection once, reduce secondary PCR verification and does not need PCR amplification subsequent treatment, greatly save time and reagent material cost and improve the working efficiency.
And (3) verification experiment: the 78 samples were reviewed according to the well-recognized fluorescent quantitative PCR method with the best detection accuracy, and the results were identical to those of the present invention.
Comparative example 1, the primers corresponding to GCR were changed to the following primer pairs:
the upstream primer GCR-F is 5' -TGCATCCGTGAAGAGAATGGTGTT,
a downstream primer GCR-R is 5' -TGCATCCGTGAAGAGAATGGTATGC;
the rest is equivalent to example 3.
As a result, the minimal amounts of five viruses including GCR, GAR, TGEV, PEDV and PCV2 detected in the multiplex PCR reaction were 5 × 103、5×102、5×101、5×102、5×101And 5 × 101copies。
Comparative example 2, the primers corresponding to PEDV were changed to the following primer pairs:
the upstream primer PEDV-F is 5' -CACGGCGACTACTCAGCTG,
the downstream primer PEDV-R is 5' -CTTCTTTAGCAACCCAGAA;
the rest is equivalent to example 3.
As a result, the minimal amounts of five viruses including GCR, GAR, TGEV, PEDV and PCV2 detected in the multiplex PCR reaction were 5 × 101、5×102、5×101、5×101、5×102And 5 × 101copies。
Comparative example 3, the primer corresponding to PCV2 was changed to the following primer pair:
the upstream primer PCV2-F: 5' -GAAGCGGACCCCAACCAC,
the downstream primer PCV2-R is 5' -ACCCAGGTGGCCCCACAAT;
the rest is equivalent to example 3.
As a result, the minimal amounts of five viruses including GCR, GAR, TGEV, PEDV and PCV2 detected in the multiplex PCR reaction were 5 × 101、5×101、5×102、5×101、5×101And 5 × 102copies。
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
<110> Zhejiang university of science and engineering
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<223> PEDV Standard
<400>11
aacacggcga ctactcagct gtgagtaatc cgagtgcggt tctcacagat agtgagaaag 60
tgcttcattt agtctaaaca gaaactttat ggcttctgtc agctttcagg atcgtggccg 120
caaacgggtg ccattatccc tctatgcccc tcttagggtt actaatgaca aacccctttc 180
taaggtactc gcaaacaacg ctgtacccac taataagggg aataaggacc agcaaattgg 240
atactggaat gagcaaattc gctggcgcat gcgccgtggt gagcgaattg aacaaccttc 300
caattggcat ttctactacc tcggaacagg acctcacgcc gacctccgtt ataggactcg 360
tactgagggt gttttctggg ttgctaaaga aggc 394
<210>12
<211>319
<212>DNA
<213> Artificial sequence
<220>
<223> TGEV Standard substance
<400>12
gtggtgttag gtgattattt tcctactgta caaccttggt ttaattgcat tcgcaatgat 60
agtaatgacc tttatgttac actggaaaat cttaaagcat tgtattggga ttatgctaca 120
gaaaatatca cttggaatca cagacaacgg ttaaacgtag tcgttaatgg atacccatac 180
tccatcacag ttacaacaac ccgcaatttt aattctgctg aaggtgctat tatatgcatt 240
tgtaagggct caccacctac taccaccaca gaatctagtt tgacttgcaa ttggggtagt 300
gagtgcaggt taaaccata 319
<210>13
<211>242
<212>DNA
<213> Artificial sequence
<220>
<223> GAR Standard substance
<400>13
tatgcaatac cagtaggacc agtatttcca ccgggtatga attggacgga attgattacc 60
aattattcac cttcaagaga agataacttg caacgtgttt ttacagtagc ttccattaga 120
agcatgttga ttaagtgagg actaggctaa ctacctggta tccgatctta accaacatgt 180
agctatgtca agtcaatcag actctacaag taagggtatg atttcatact cgctacgtag 240
ag 242
<210>14
<211>126
<212>DNA
<213> Artificial sequence
<220>
<223> GCR standard substance
<400>14
tgttgcatcc gtgaagagaa tggtgttgta aagaagctag agatctaaca aatctctatg 60
tggactacgc accatgtagc atgattcacg aatgggttta gtccatgctt gcgtaggggc 120
aaatgc 126
<210>15
<211>508
<212>DNA
<213> Artificial sequence
<220>
<223> PCV2 standard substance
<400>15
aagaagcgga ccccaaccac acaaaaggtg ggtgttcacg ttgaataatc cttccgagga 60
cgagcgcaag aaaatacggg agcttccaat ctcccttttt gattatttta ttgttggcga 120
ggagggtaat gaggaaggac gaacacccca cctccagggg ttcgctaatt ttgtgaagaa 180
gcaaacattt aataaagtga aatggtattt cggtgcccgc tgccacatcg agaaagcgaa 240
aggaactgat cagcagaata aagaatattg cagtaaagaa ggcaacttac tgattgaatg 300
tggagctcct agatctcagg gacaacggag tgacctgtct actgctgtga gtaccttgct 360
ggagagcggg agtctggtga ccgttgcaga gcagcaccct gtaacgtttg tcagaaattt 420
ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg cagaagcgtg attggaagac 480
taatgtacac gtcattgtag ggccacct 508
Claims (7)
1. Five pig diarrhea virus multiple PCR rapid diagnosis kits, which is characterized in that:
comprises the following 5 pairs of PCR primers:
2. the five porcine diarrhea virus multiplex PCR rapid diagnostic kit according to claim 1, characterized in that the kit comprises:
a) PCR reaction solution, b) PCR standard, c) positive control, d) negative control and e) primer.
3. The five porcine diarrhea virus multiplex PCR rapid diagnostic kit according to claim 2, characterized in that:
the PCR standard consists of the following 5 standards: PEDV standard, TGEV standard, GAR standard, GCR standard, PCV2 standard.
4. The five porcine diarrhea virus multiplex PCR rapid diagnostic kit according to claim 2, characterized in that:
the PCR reaction solution comprises a PCR reaction buffer solution and heat-resistant DNA polymerase;
the PCR reaction buffer contains buffer solution, magnesium chloride and a deoxyribonucleotide triphosphate mixture.
5. The five porcine diarrhea virus multiplex PCR rapid diagnostic kit according to claim 2, characterized in that:
the positive control contains positive plasmid mixture of five viruses of TGEV, GAR, GCR, PEDV and PCV 2;
the negative control substance is sterilized diethyl pyrocarbonate treated water.
6. The five porcine diarrhea virus multiplex PCR rapid diagnostic kit according to claim 2, characterized in that:
the upstream and downstream primers of the specific primers for each virus were packaged in the same tube.
7. The five porcine diarrhea virus multiplex PCR rapid diagnostic kit according to claim 2, characterized in that:
the kit is stored at-20 ℃ in the dark.
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| CN107955839A (en) * | 2017-12-13 | 2018-04-24 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of 3 type of porcine circovirus 2 type and circovirus |
| CN108715906A (en) * | 2018-06-04 | 2018-10-30 | 广西大学 | Five kinds of pig enterovirus multiple RT-PCR quick detection kits and its application |
| CN109097499A (en) * | 2018-09-19 | 2018-12-28 | 浙江农林大学 | It is a kind of for detecting the dual fluorescence quantification RT-PCR detection kit of PEDV-TGEV nucleic acid |
| CN114807437B (en) * | 2022-04-06 | 2023-03-31 | 南京农业大学 | Quadruple fluorescent quantitative PCR detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus |
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| CN104561368A (en) * | 2014-09-30 | 2015-04-29 | 浙江理工大学 | Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses |
| CN105385683A (en) * | 2015-06-01 | 2016-03-09 | 蒋春燕 | Multiplex RT-PCR detection kit for porcine epidemic diarrhea viruses |
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| CN104561368A (en) * | 2014-09-30 | 2015-04-29 | 浙江理工大学 | Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses |
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