CN103225001B

CN103225001B - Porcine circovirus type 2 rapid typing detection kit

Info

Publication number: CN103225001B
Application number: CN201310162390.8A
Authority: CN
Inventors: 杨毅; 忻星; 王乃东; 邓治邦; 薛立群; 湛洋; 首易君
Original assignee: Individual
Current assignee: Hunan Paizhi Biotechnology Co ltd
Priority date: 2013-05-06
Filing date: 2013-05-06
Publication date: 2014-12-31
Anticipated expiration: 2033-05-06
Also published as: CN103225001A

Abstract

The invention relates to a porcine circovirus type 2 rapid typing detection kit. The kit belongs to the field of biological detection, is a kit based on a SMART determination system method, solves the technical problems of complex operation procedure, higher experimental skill requirement, longer experimental period and the like of a porcine circovirus type 2 (PCV 2a, 2b, 2c, 2d and the like) typing detection method, and can provide a rapid and accurate molecular diagnosis technical product for porcine circovirus type 2 typing. The invention can be used for the systematic diagnosis of the rapid typing of the porcine circovirus type 2 to guide the prevention and treatment of the disease, can realize the detection of various sequence mutations of the virus, and is beneficial to the molecular epidemiological investigation of the porcine circovirus, the meat product quarantine, the virus monitoring of feed and water quality of a pig farm, and the like.

Description

A kind of porcine circovirus 2 type rapid typing detection reagent box

Technical field

The present invention relates to a kind of porcine circovirus 2 type rapid typing detection reagent box, belong to field of biological detection.

Background technology

Pig circular ring virus (porcine circovirus type2, PCV-2) belongs to PCV-II section, and diameter is only 17nm, without cyst membrane, and icosahedral symmetry, virus covalently closed circular Single-stranded DNA virus.PCV-2 causes multisystemic exhaustion syndrome (postweaning multisystemic wasting syndrome after weaned piglet, PMWS) main pathogen, this disease from 1991 in Canadian swinery after the first explosion, be widely current in world community swinery, cause huge financial loss to world's pig industry.Normal and porcine reproductive and respiratory syndrome virus (the porcine reproductive and respiratory syndrome virus of PCV-2, PRRSV), pig parvoviral (porcine parvovirus, PPV), PRV (Pseudorabies virus) (pseudorabies virus, PRV), haemophilus parasuis (Haemophilus parasuis, HPs), actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, and pig pasteurellosis bacillus (swine pasteurellosis App), concurrent or the secondary infection of many cause of diseases such as Sp), add its difficulty of prevention and cure.Except causing PMWS, PCV-2 also can cause growing and fattening pigs dermatitis and nephrotic syndrome (porcine dermatis and nephropathy syndrome, PDNS), porcine respiratory syndrome (porcine respiratory disease complex, PRDC), Hypertrophic necrotizing pneumonia (proliferativenecrotising pneumonia, PNP), immunosuppression in the syndromes such as the dysentery of newborn piglet and pig body, thus be difficult to carry out effective pharmacological agent, add the very high and existence of avirulent PCV-1 of homology with it, the Accurate Diagnosis infected for PCV-2 undoubtedly and effectively prevention and control add difficulty.At the beginning of 2000, PCV-2 has become worldwide popular, causes huge financial loss to each Swine Production big country of the world (North and South America, Europe, and Asia).Many data show, although the infection positive rate of PCV-2 in China each department swinery is not quite similar, its extensive existence in China swinery has been undisputable fact, form significant threat to the health of swinery.In current China swinery, PCV-2 presents polygene type polyinfection, and different genotype strain coexists, and that has reported comprises PCV2A, PCV2B, PCV2D, PCV2C.4 strain different genotype strains, their core area sequence does not change, and sudden change is present in outside surface (Cap protein amino acid), and there is some difference for the antigenicity of therefore different genotype strain.

Pig circular ring virus is divided into 2 serotypes, PCV1 and PCV2.Wherein PCV1 type does not cause clinical symptom, is extensively present in each organ-tissue of normal pig body and pig source cell.PCV2 all has pathogenic to the family pig at various age and wild boar.Corresponding with serum group system, pig circular ring virus genome is also divided into 2 kinds of genotype, i.e. PCV1 type genome and PCV2 type genome.PCV2 type full-length genome is 1766bp, 1767bp or l768bp, usually containing 11 ORF, wherein ORFl, ORF5, ORF7 and ORF10 are on viral chain, and ORF2, ORF3, ORF4, ORF6, ORF8, ORF9 and ORF11 are on complementary strand, these gene expression are overlapping genes, thus take full advantage of the limited genetic material of virus.ORFl and ORF2 is 2 main open reading frame, the Rep ' albumen that coding is relevant with virus replication respectively and the capsid protein (Cap) of virus.In ori (Ori) the stem ring sequence that the region intermediate of ORF1 and ORF2 is viral DNA, synthesize at the albumen of virus, DNA self-replication and progeny virus play a significant role in producing.Find the genome analysis of PCV2, the genomic homology of all PCV2 strain isolateds all more than 90%, but only has 68% ~ 79% with the nucleotide homology of PCVl.79.5% and 82% is about respectively to the homology copying relevant DNA replication dna initiator and replicative enzyme encoding gene (rep) sequence between PCV1 and PCV2; But there is larger difference in its capsid protein encoding gene (cap), homology only has an appointment 62%.

The sorting technique of PCV2a, PCV2b, PCV2c tri-kinds of gene hypotypes that is divided into by PCV2 that the Porcine circovirus desease council of European Union proposes is widely accepted, the fundamental principle of its somatotype be full genome genetic distance be greater than 0.02 and ORF2 gene genetic distance be greater than 0.035, wherein PCV2a and PCV2b is the main genotypes existed at present.The relation of genotype and virulence also becomes the focus that PCV2 studies at present, Grau-Roma etc. find that the PCV2 be separated in the swinery that PMWS occurs belongs to PCV2b (in original text called after genotype1) more, and belong to PCV2a (original text called after genotype2) from healthy swinery with the PCV2 having the pig of symptom of slightly becoming thin to be separated to more, there is the strain of different virulence in result prompting PCV2. and gene type may to judging that disease popularity situation has very great help.And phylogenetic analysis display, PCV2a type strain is early than the strain of PCV2b type on time of occurrence, and many countries also find constantly breaking out along with PMWS, has occurred the phenomenon changed to PCV2b genotype by PCV2a.In addition have people also to find to be separated to multiple PCV2 strain isolated in same pig body, these strain isolateds of order adhere to different genotype separately, and this phenomenon prompting gene recombination may play an important role in PCV2 genetic evolution.According to the sorting technique of the Porcine circovirus desease council of European Union. the current PCV2 strain isolated of China belongs to PCV2b hypotype more.

The Detection and diagnosis of pig PCV-2 is the prerequisite of epidemic prevention and control, and the somatotype Detection and diagnosis of pig PCV-2 has guiding significance to the research of follow up vaccine, proper use of and treatment etc.But the acceleration due to PCV-2 pathogenic agent suddenlys change and the feature of polygene type PCV-2 polyinfection in pig farm, traditional medical science detection means (regular-PCR, ELISA etc.) cannot detect mutant and the classification system Detection and diagnosis of novel pathogenic agent, and the develop rapidly of Protocols in Molecular Biology and fluorescent quantitation detection technique, for the quantitative and qualitative analysis detection of novel pathogenic agent and mutant thereof provides possibility.

Current real-time quantitative PCR (real time PCR; qPCR) PCV2 can be realized to measure fast; existing academic article and patent report; PCV2 real-time quantitative PCR reaction system often comprises four components: (1) reaction solution (master mix); usually damping fluid is had; magnesium ion, enzyme, and dNTP etc.; (2) tested viral nucleic acid karyomit(e) (target DNA), (3) primer (primers), (4) probe (probe), the most frequently used probe is TaqMan probe.But in PCV2 real-time quantitative PCR reaction system detection system, because the specificity of primer is inadequate, so need probe to ensure atopic.Probe needs fluorescent mark usually, and namely probe oligonucleotide (oligo nucleotide) needs coupling fluorophor (fluorophore), and a quenching group (quencher).From synthesis angle, the difficulty of fluorescent probe synthesis is larger than primer synthesis, so probe price is commercially often higher than primer ten times.

Summary of the invention

The object of this invention is to provide a kind of simple and rapid porcine circovirus 2 type rapid typing detection reagent box, this detection kit is the test kit measuring system method based on SMART.

A kind of porcine circovirus 2 type rapid typing detection reagent box, this detection kit is the test kit measuring system method based on SMART, comprises following three pairs of primers:

In SMART mensuration system, because the specificity of primer is extremely strong, so need not can reach the requirement of the high degree of specificity of reaction by probe, SMART detection system comprises and has high fluorescent, high atopic EvaGreen and can the specific CrystalTaq of intensified response.The PCV2a that our SMART detection system comprises, in PCV2b, PCV2c primer sequence, reverse primer is that three kinds of genotype all have, and forward primer can distinguish PCV2a, PCV2b, PCV2c.As shown in Fig. 1, Fig. 2, Fig. 3, SMART detection system does not have cross reaction between three kinds of genotype, and with common system (SYBR GreenER Super Mix, Life Technologies, Carlsbad, California, USA) cross reaction in various degree can be there is.So conventional system must use expensive probe to ensure the specificity of reaction.That is, the more simple system of SMART System application reaches the object of complex system (conventional system).And the conventional system of application probe also has another problem: whether its detection system (experiment reagent box) needs the sample comprising positive control, effective with the reaction solution of test kit, primer and probe.In above-mentioned three kinds of compositions, any one lost efficacy and reaction will be made to lose efficacy.Positive control sample normally artificial nucleic acid simulates actual detection target.But this simulation reality detects target once pollute reactive system, just cannot distinguish positive control sample and actual sample (because fluorescent signal is all the same).If must distinguish, just need to open each reaction reagent, do further inspection, but this step can increase the chance of pollution.And in our SMART detection system, artificial simulation nucleic acid and actual target DNA adopt same primer to increase, but length is different, so there is same PCR amplified curve, but artificial simulation nucleic acid on melting curve and actual target DNA different.According to this point, can accurately judge to pollute or real positive signal (Fig. 4).

Accompanying drawing explanation

Fig. 1 is that PCV2a SMART PCR reacts and the detection of SYBR GreenER regular-PCR is compared;

Fig. 2 is that PCV2b SMART PCR reacts and the detection of SYBR GreenER regular-PCR is compared;

Fig. 3 is that PCV2c SMART PCR reacts and the detection of SYBR GreenER regular-PCR is compared;

The behave Tm of mold board and actual template of Fig. 4 compares.

Embodiment

Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.

Embodiment 1, at the qualitative PCV of real-time SMART fluorescent quantitation.

Step 1: in PCR real time reaction pipe a, add 10 μ l2X SMART Master Mix (comprising EvaGreen fluorescence dye, SMART buffering system, SMART Taq, dNTP, magnesium ion), 500nM PCV2a forward primer and PCV2a reverse primer, with 1 μ l determined nucleic acid sample, with pure water, total reaction volume is added to 20 μ l.

Step 2: in PCR real time reaction pipe b, adds 10 μ l2X SMART Master Mix, 500nM PCV2b forward primer and PCV2b reverse primer, and 1 μ l determined nucleic acid sample, with pure water, total reaction volume is added to 20 μ l.

Step 3: in PCR real time reaction pipe c, adds 10 μ l2X SMART Master Mix, 500nM PCV2c forward primer and PCV2c reverse primer, and 1 μ l determined nucleic acid sample, with pure water, total reaction volume is added to 20 μ l.

Step 4: above three pipes are placed on ABI7500(or ABI7900, or BioRad, Bioer, Roche, the instrument of identity function) innerly carry out PCR in real time amplification.The temperature condition of reaction is:

95 DEG C of warm starts in 2 minutes,

Circulate 40 times between 95 DEG C of 15s and 65 DEG C 30s.

Fluorescence is detected at 65 DEG C.

Embodiment 2, in quantitative real-time PCR, SMART has more high specific

The present invention compares SMART PCR and to react and with the specificity of the SYBR GreenER regular-PCR that is representative.Result as shown in Figure 1.The upper width of Fig. 1 shows in 10 μ LSMART PCR reaction systems containing 250nM, be used for detecting the positive and negative primer (table 1 shown in) of PCV2a, with the PCV2a being respectively 1,000,000 copies, or PCV2b, or PCV2c, or the SYBR GreenER that the lower width of NTC. Fig. 1 shows 10 μ L is the positive and negative primer (shown in table 1) being used for detecting PCV2a in the common response system of representative containing 250nM concentration, with the PCV2a being respectively 1,000,000 copies, or PCV2b, or PCV2c, or NTC.

Table 1

Fig. 1 clearly shows, SMART PCR atopic, and does not find cross reaction, and SYBR GreenER is the common response system of representative and PCV2b template cross reaction.This example describes the superiority utilizing SMART PCR reaction detection PCV2a.

Embodiment 3, in quantitative real-time PCR, SMART has higher specificity

The present inventor again compares SMART PCR and to react and with the specificity of the SYBR GreenER regular-PCR that is representative.Result as shown in Figure 2.The upper width of Fig. 2 to show in the SMART PCR reaction system of 10 μ L containing 250nM concentration, is used for detecting the positive and negative primer (shown in table 1) of PCV2b, and is respectively the PCV2a of 1,000,000 copies, or PCV2b, or PCV2c, or NTC.The lower width of Fig. 2 show 10 μ LSYBR GreenER be in the common response system of representative containing 250nM, be used for detecting the positive and negative primer (shown in table 1) of PCV2b, and be respectively the PCV2a of 1,000,000 copies, or PCV2b, or PCV2c, or NTC.

Fig. 2 clearly shows, and SMART PCR reacts and specificity, and does not have cross reaction, and SYBR GreenER is common response system and the PCV2a template of representative, and PCV2c template has cross reaction.This example again illustrates SMART PCR and reacts the superiority on mensuration PCV2b.

Embodiment 4, in quantitative real-time PCR, SMART has higher specificity

The present inventor again compares SMART PCR and to react and with the specificity of the SYBR GreenER regular-PCR that is representative.Result as shown in Figure 3.The upper width of Fig. 3 to show in the SMART PCR reaction system of 10 μ L containing 250nm concentration, is used for detecting the positive and negative primer (shown in table 1) of PCV2c, and is respectively the PCV2a of 1,000,000 copies, or PCV2b, or PCV2d, or NTC.The SYBR GreenER that the lower width of Fig. 3 shows 10 μ L is containing 250nM concentration in the common response system of representative, be used for detecting the positive and negative primer (table 1 shown in) of PCV2c, and be respectively the PCV2a of 1,000,000 copies, or PCV2b, or PCV2c, or NTC.

Fig. 3 clearly shows, and SMART PCR reacts and specificity, do not have cross reaction, and SYBR GreenER is common response system and the PCV2b template of representative, and PCV2c template has cross reaction.This example again illustrates SMART PCR and reacts the superiority on mensuration PCV2c.

Embodiment 5, can distinguish actual sample and artificial control sequence in real time fluorescent quantitative SMART PCR method.

In SMART mensuration system of the present invention, artificial simulation nucleic acid and actual target DNA adopt same primer, but intermediate sequence is different (as shown in table 2, naturally the sequence found is TTGACA between two primers, the intermediate sequence of artificial simulated templates is TGCGCGCA, two are amplified product due to sequence difference, and the temperature of melting has difference).PCR amplified curve is identical, but artificial simulation nucleic acid is different, as shown in table 2 with the target DNA sequence dna of reality on melting curve.As shown in Fig. 4 melting curve, people's mold board is different with the Tm of actual template.Although this detected result shows, people's mold board Tm is higher than actual template Tm, and in actual applications, people's mold board Tm also can lower than the Tm of actual template.

Table 2: actual template sequence and artificial template sequence

(below the sequence that primer pair is answered, adding thick stick)

Claims

1. a porcine circovirus 2 type rapid typing detection reagent box, is characterized in that, this detection kit is the test kit measuring system method based on SMART, comprises following three pairs of primers:

Also comprise: intermediate sequence is the artificial simulated templates of TGCGCGCA.