CN104593526B

CN104593526B - Method and primer group for detecting porcine circovirus

Info

Publication number: CN104593526B
Application number: CN201510060875.5A
Authority: CN
Inventors: 傅生芳; 余黎; 周旭; 寇桂英; 安红; 包红
Original assignee: Lanzhou Institute Of Biological Products Co ltd
Current assignee: Lanzhou Institute Of Biological Products Co ltd
Priority date: 2015-02-05
Filing date: 2015-02-05
Publication date: 2017-10-27
Anticipated expiration: 2035-02-05
Also published as: CN104593526A

Abstract

The invention provides a universal semi-nested PCR primer for type I and type II PCV and a specific nested PCR primer for various PCV, establishes a porcine circovirus detection method, and is proved to be rapid, specific and effective by sequence determination.

Description

Method and primer group for detecting porcine circovirus

Technical Field

The invention relates to a method for detecting porcine circovirus, in particular to a method for detecting porcine circovirus in rotavirus vaccine.

Background

The porcine circovirus belongs to the genus circovirus of the family circovirus, the virus particle size is 14-25 nm, the average diameter is 17nm, the virus particle is a symmetrical icosahedron without a capsule membrane, and the genome is single-stranded circular DNA. The suspension density in the tissue is 1.37cm³The sedimentation coefficient is 52S, which is the smallest animal virus found at present. PCV has strong resistance to the outside, and the virus is insensitive to chloroform and does not agglutinate red blood cells of various animals such as cattle, sheep, pigs, chickens and the like and human beings.

PCV is classified into PCV-1 and PCV-2 according to differences in pathogenicity, antigenicity, and nucleic acid sequence. PCV-1 was nonpathogenic, first discovered in 1974 by Tischer, a German scholarer, from multiple strains of serially passaged PK-15 cells, and it was demonstrated in 1982 that the virus originated from porcine kidney tissue from which PK-15 cells were originally prepared. Later, the virus is proved to be only a common virus capable of infecting pigs and cannot cause harm to the infected pigs. PCV-2 is pathogenic and causes pigs to exhibit a variety of clinical symptoms.

On day 22/3 2010, the FDA announces suspension of use of the rotavirus vaccine Rotarix produced by GSK corporation because of contamination of the vaccine with porcine circovirus type 1 (PCV1) DNA. This conclusion was reached in the laboratory of Eric Delwart using a new technique called Deep sequencing. However, deep sequencing also has certain disadvantages in application, which mainly includes complex detection method, high cost and the like.

At the same time, subsequent testing by GSK and FDA confirmed the presence of this DNA in the vaccine. On 6.5.2010, the FDA announced that RotaTeq (produced by Merck) contains DNA fragments of porcine circovirus type I (PCV1) and porcine circovirus type II (PCV 2). Both vaccines inherently have excellent safety records. To date, there is no evidence that the presence of PCV in these 2 vaccines can cause infection in humans, but the presence of pathogens of this animal origin will eventually be at risk, suggesting importance for the control of vaccine quality.

Therefore, how to effectively detect the porcine circovirus in the rotavirus vaccine so as to improve the purity of the vaccine becomes an urgent problem to be solved.

Disclosure of Invention

Aiming at the problems, the invention respectively designs the universal semi-nested PCR primers of the PCV of types I and II and the specific nested PCR primers of each PCV, establishes a detection method of porcine circovirus, and is proved to be quick, specific and effective by sequence determination.

One aspect of the present invention provides a method for detecting porcine circovirus, wherein the porcine circovirus includes type I and type II viruses, the method comprising:

extracting DNA from a sample, and carrying out first PCR; and

performing a second PCR on the amplification product obtained by the first PCR, the present application provides the following primer sets:

the primers used in the first PCR include a first primer set, a second primer set and a third primer set,

the primers used in the second PCR include a fourth primer set, a fifth primer set and a sixth primer set,

wherein,

the first primer group comprises an upstream primer with a sequence shown as SEQ NO. 1 and a downstream primer with a sequence shown as SEQ NO. 2,

the second primer group comprises an upstream primer with a sequence shown as SEQ NO. 3 and a downstream primer with a sequence shown as SEQ NO. 4,

the third primer group comprises an upstream primer with a sequence shown as SEQ NO. 5 and a downstream primer with a sequence shown as SEQ NO. 6,

the fourth primer group comprises an upstream primer with a sequence shown as SEQ NO. 1 and a downstream primer with a sequence shown as SEQ NO. 7,

the fifth primer group comprises an upstream primer with a sequence shown as SEQ NO. 8 and a downstream primer with a sequence shown as SEQ NO. 9,

the sixth primer group comprises an upstream primer with a sequence shown as SEQ NO. 10 and a downstream primer with a sequence shown as SEQ NO. 11.

Wherein, the detected sample is rotavirus oral live vaccine, 3-valent rotavirus recombined vaccine, calf serum, a culture medium, pancreatin for cell digestion, pancreatin for virus digestion and/or Vero cells.

Wherein the method further comprises performing qualitative analysis on the product after the second PCR amplification. Qualitative analysis included visualization of the product after the second PCR amplification by gel electrophoresis.

The invention also provides a primer group for detecting the porcine circovirus, wherein the primer group is a universal primer group which can detect the porcine circovirus including the type I and/or the type II virus, the primer group comprises an upstream primer and a downstream primer, and the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 1 and a downstream primer with a sequence shown as SEQ NO. 2.

The invention also provides a primer group for detecting the porcine circovirus, wherein the primer group is a universal primer group which can detect the porcine circovirus including the type I and/or the type II virus, the primer group comprises an upstream primer and a downstream primer, and the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 1 and a downstream primer with a sequence shown as SEQ NO. 7.

The other aspect of the invention provides a primer group for detecting porcine circovirus, wherein the primer group can detect porcine circovirus type I, the primer group comprises an upstream primer and a downstream primer, and the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 5 and a downstream primer with a sequence shown as SEQ NO. 6.

Another aspect of the invention is to provide a primer set for detecting porcine circovirus, wherein the primer set can detect porcine circovirus type I, the primer set comprises an upstream primer and a downstream primer, and the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 10 and a downstream primer with a sequence shown as SEQ NO. 11.

The other aspect of the invention provides a primer group for detecting porcine circovirus, wherein the primer group can detect the porcine circovirus II, the primer group comprises an upstream primer and a downstream primer, and the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 3 and a downstream primer with a sequence shown as SEQ NO. 4.

The other aspect of the invention provides a primer group for detecting porcine circovirus, wherein the primer group can detect the porcine circovirus II, the primer group comprises an upstream primer and a downstream primer, and the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 8 and a downstream primer with a sequence shown as SEQ NO. 9.

The invention has the following characteristics:

1. the universal primers I and II are designed according to the conserved regions of PCV1 and PCV2, and the PCV1 fragment synthesized by the semi-nested PCR primer pair and the PCV2 cell culture can amplify a specific fragment of 260 bp. After sequence determination and alignment, the fragment has high homology with other PCV1 strains and PCV-II strains (the homology of the fragment amplified by taking PCV1 as a template and PCV1 strains is more than 96 percent, and the homology of the fragment amplified by taking PCV2 cell culture as a template and PCV strain II is more than 97 percent).

2. On the other hand, the applicant of the invention designs a PCV-I specific nested PCR primer, amplifies the synthesized PCV-I fragment, and both the outer primer and the inner primer can amplify target fragments with corresponding expected sizes, and after sequencing and sequence comparison, the primers have high gene homology (over 99 percent) with other PCV1 strains.

3. On the other hand, the applicant of the invention designs a nested PCR primer specific to PCV2, amplifies a PCV-II fragment, and after sequencing the obtained specific fragment of 500bp, the homology with other PCV2 strains is high (more than 98%).

4. Another aspect of the present invention is to provide an effective method for detecting PCV-I and PCV-II in rotavirus vaccines and other auxiliary products.

Drawings

The invention will be further explained with reference to the accompanying drawings, in which:

FIG. 1 is a photograph of agarose gel electrophoresis analysis of an amplification product of PCV-I and PCV-II universal primers using a synthesized PCV-I fragment positive recombinant plasmid as a template;

FIG. 2 is a photograph of agarose gel electrophoresis analysis of an amplification product of a PCV-I specific primer using a synthesized PCV-I fragment-positive recombinant plasmid as a template;

FIG. 3 is a photograph of agarose gel electrophoresis analysis of PCV-I and PCV-II universal primer amplification products using PCV-II cell culture as a template;

FIG. 4 is a photograph of an agarose gel electrophoresis analysis of an amplification product of a PCV-II specific primer using a PCV-II cell culture as a template;

FIG. 5 is a photograph of agarose gel electrophoresis analysis of the PCV-I and PCV-II universal primer amplification products using various rotavirus vaccines and related components as templates;

FIG. 6 is a photograph of agarose gel electrophoresis analysis of the amplification products of PCV-I specific primers using various rotavirus vaccines and related components as templates;

FIG. 7 is a photograph of agarose gel electrophoresis analysis of the PCV-II specific primer amplification products using various rotavirus vaccines and related components as templates.

In the following examples, unless otherwise specified, all methods are conventional. The experimental materials or biological agents used in the following examples are, unless otherwise specified, conventional reagents available on the market. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

Detailed Description

The following preferred embodiments are supplementary to the present invention or the present invention, but are not intended to limit the contents of the present invention. The following primers and genes were synthesized by Shanghai Biotech services Ltd.

1 detecting the sample

Due to the absence of PCV-I virus, a partial gene fragment of PCV-I virus was designed and synthesized. The partial gene fragment (full length 730bp, 81 bp-810 bp) of PCV-I virus designed and synthesized according to the sequence registered in Genebank is shown as SEQ NO: 12. The sample of porcine circovirus type II was a culture of porcine kidney cell virus cultured by the second laboratory of Limited liability company of the Lanzhou Biopreparation institute. The virus and cell lines were provided by the Lanzhou veterinary institute. The rotavirus oral live vaccine is provided by a vaccine room of a Limited liability company of Lanzhou biological product research institute, and the 3-valent rotavirus gene recombined vaccine is provided by a second research room of the Limited liability company of the Lanzhou biological product research institute. All test samples were concentrated to at least 100-fold using a concentration tube, except for 10-fold concentration of newborn bovine serum.

2 reagent

DNA extraction reagent DNA zol was purchased from Invitrogen corporation, Ex Taq enzyme and DNA Marker are products of Takara corporation; the Agarose Gel DNA recovery kit was purchased from QIGEN. Newborn bovine serum was purchased from glorious biotechnology, Inc., Lanzhou, MEM was purchased from GIBCO, digestive viruses were purchased from Sigma using trypsin, digestive cells were purchased from BD using trypsin, and Vivaspin15 concentration tubes were purchased from Sartorius.

3 design and synthesis of primers and Gene fragments

The universal semi-nested PCR primers for PCV1 and PCV2 and the specific nested PCR primers for each type were designed by means of Primer 5.0 based on the sequences of GeneBank accession numbers JN133301.1, GU799575.1, GQ449671.1 and EF52452.1, respectively, with the expected amplified fragment lengths of 260bp, 434bp and 500bp, respectively, and the Primer sequences are as shown in Table 1 below:

TABLE 1 primer sequences

4 extraction of DNA

Extraction was performed using the DNA zol reagent protocol. The extracted samples include, for example, a type II porcine circovirus sample (obtained by culturing PCV-II in PK cells (supplied from Lanzhou veterinary institute) by a second laboratory), a PCV-I type virus recombinant plasmid (trusted Shanghai bioengineering, Inc.), calf serum (obtained from Lanzhou RongLei-Lexan Biotech, Inc.), MEM medium (obtained from GBICO), trypsin for digestive cells (obtained from BD), trypsin for digestive viruses (obtained from SIGMA), Vero cells (obtained from ATCC), oral rotavirus live vaccine (commercially available vaccine from Lanzhou Biotech, Inc., Rotavirus LD strain (2 type strain reassorted from the second laboratory of Lanzhou Biotech, Inc.), rotavirus LS strain (3 type reassorted from Lanzhou Biotech, Inc.), and the like, Rotavirus LH strain (type 4 strain reassorted by the second institute of limited responsibility of the lanzhou biological products institute), 3-valent rotavirus gene reassorted vaccine (vaccine developed by the second institute of limited responsibility of the lanzhou biological products institute).

5 nested PCR

5.1 first PCR

The DNA extracted in the previous step was aspirated in 4. mu.l each as a template to amplify fragments PCS2A3, S2A2 and P3P4, respectively. Mu.l each of the upstream sense primers PCS2(10pM), S2(10pM) and P3(10pM), 1. mu.l each of the downstream antisense primers PCA3(10pM), A2(10pM) and P4(10pM), 1. mu.l each of dNTPs (10 mM each), 5. mu.l each of 10 XEx taq buffer, 37.75. mu.l each of sterilized distilled water, and 0.25. mu.l each of Ex taq were added. A50. mu.l reaction was subjected to a first amplification in a PCR amplification apparatus. The procedure is as follows: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, denaturation at 52 ℃ for 30s, and denaturation at 72 ℃ for 30s, followed by 30 cycles and elongation at 72 ℃ for 5 min.

5.2 second PCR

Mu.l of the first PCR product was used as a template to amplify fragments PCS2A2, S3A3 and P5P6, respectively. The upstream sense primers PCS2(10pM), S3(10pM) and P5(10pM) were added, the downstream antisense primers PCA2(10pM), A3(10pM) and P6(10pM) were added in an amount of 1. mu.l each, dNTP (10 mM each) was added in an amount of 1. mu.l each, 10 XEx taq buffer was added in an amount of 5. mu.l each, distilled sterilized water was added in an amount of 39.75. mu.l each, and Ex taq was added in an amount of 0.25. mu.l each. A50. mu.l reaction was subjected to a second amplification in a PCR amplification apparatus. The procedure is as follows: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, denaturation at 55 ℃ for 30s, and denaturation at 72 ℃ for 30s, followed by 30 cycles, and elongation at 72 ℃ for 5 min. Finally, 5. mu.l of the nested PCR (second PCR) product was subjected to electrophoresis on a 2% agarose gel.

6 PCR product sequencing

After the second PCR product was recovered by Agarose Gel DNA recovery kit (QIAGEN), 10. mu.l of the second PCR product was sequenced by Shanghai Biotech service Co., Ltd, and the sequencing primers were PCS2, S3 and P5, respectively.

7Sequence is the same asSource analysis

Using DNAstar software.

8Results

8.1RT-PCR results

The synthesized PCV-I fragment (the sequence is shown as SEQ NO: 12) positive recombinant plasmid is used as a template, and the expected target fragments of 537bp and 260bp can be respectively amplified by the two types of universal nested primers after 2 times of PCR amplification. The results are shown in FIG. 1. Wherein, the band 1-1 is a product band after the first amplification of the PCV-I recombinant plasmid, the band 1-2 is a product band after the second amplification of the PCV-I recombinant plasmid, and the band 1-3 is a 100bp ladder.

Taking the positive recombinant plasmid of the synthesized PCV-I fragment (the sequence is shown as SEQ NO: 12) as a template, and amplifying DNA target fragments of 647bp and 434bp respectively by using the type I specific nested primer through 2 times of PCR; the results are shown in FIG. 2. Wherein, the band 2-1 is 100bp ladder, the band 2-2 is a product band after the first amplification of the PCV-I recombinant plasmid, and the band 2-3 is a product band after the second amplification of the PCV-I recombinant plasmid.

DNA of PCV-II pig kidney cell culture is taken as a template, after nested PCR amplification, a specific band of 260bp and 500bp is respectively amplified by two types of universal primers and II type specific primers and is consistent with the size of an expected target fragment. FIG. 3 is a photograph showing an agarose gel electrophoresis analysis of an amplification product of PCV-I and PCV-II universal primers using PCV-II cell culture as a template, wherein lane 3-1 is a lane for the second amplification of PCV-II cell culture, and lane 3-2 is 100 bpladder. FIG. 4 is a photograph showing an agarose gel electrophoresis analysis of an amplification product of PCV-II specific primers using PCV-II cell culture as a template, wherein bands 4-1 and 4-2 are second amplification bands of PCV-II cell culture, and band 4-3 is 100 bpladder.

Under the condition that the positive control is established, no specific expected band is amplified in the rotavirus vaccine and related components to be detected. The results are shown in FIGS. 5-7.

Specifically, FIG. 5 is a photograph of agarose gel electrophoresis analysis of the amplification products of PCV-I and PCV-II universal primers using various rotavirus vaccines and related components as templates.

Wherein, the strip 5-1 is a positive control (PCV-II cell culture) twice amplification product strip; the strip 5-2 is a strip of a product obtained by twice amplification of calf serum; the band 5-3 is a band of products of two amplifications in MEM medium; the band 5-4 is the product band of the two-time amplification of the digested cells with trypsin; bands 5-5 are bands of products amplified twice with trypsin for digesting viruses; the strip 5-6 is a Vero cell twice amplification product strip; the bands 5-7 are bands of products obtained by twice amplification of oral live rotavirus vaccines; the strip 5-8 is the strip of the product of the two amplifications of 3-valent rotavirus gene reassortment seedling; bands 5-9 are 100bp Ladder; the band 5-10 is the band of the products of two amplifications of rotavirus LD strain; the bands 5-11 are bands of two amplification products of rotavirus LS strain; the bands 5-12 are bands of two amplification products of rotavirus LH strain.

Specifically, FIG. 6 is a photograph of agarose gel electrophoresis analysis of the amplification products of PCV-I specific primers using various rotavirus vaccines and related components as templates.

Wherein, the band 6-1 is a positive control (PCV-I recombinant plasmid) primary amplification product band; the band 6-2 is a positive control (PCV-I recombinant plasmid) secondary amplification product band, and the band 6-3 is a calf serum secondary amplification product band; the band 6-4 is a band of products of two amplifications in MEM medium; the band 6-5 is the product band of the two-time amplification of the digested cells with trypsin; bands 6-6 are bands of products amplified twice with trypsin for digesting viruses; the band 6-7 is the Vero cell twice amplification product band; bands 6-8 are 100bp Ladder; the bands 6-9 are bands of products obtained by twice amplification of oral live rotavirus vaccines; the band 6-10 is the band of the products of two amplifications of rotavirus LD strain; the bands 6-11 are bands of two amplification products of rotavirus LS strain; the bands 6-12 are bands of two amplification products of rotavirus LH strain; the bands 6-13 are bands of products obtained by twice amplification of 3-valent rotavirus gene reassortment seedlings.

Specifically, FIG. 7 is a photograph of agarose gel electrophoresis analysis of the PCV-II specific primer amplification products using various rotavirus vaccines and related components as templates.

Wherein, the band 7-1 is a positive control (PCV-II cell culture) secondary amplification product band; the band 7-2 is a band of a product obtained by amplifying calf serum twice; the band 7-3 is a band of products of two amplifications in MEM medium; band 7-4 is the product band of two trypsinization of the digested cells; the band 7-5 is a band of a product obtained by twice amplifying the digested virus with trypsin; the band 7-6 is the Vero cell twice amplification product band; bands 7-7 are 100bp Ladder; the bands 7-8 are bands of products obtained by twice amplification of oral live rotavirus vaccines; the bands 7-9 are bands of two amplification products of rotavirus LD strain; the bands 7-10 are bands of two amplification products of rotavirus LS strain; the bands 7-11 are bands of two amplification products of rotavirus LH strain; the bands 7-12 are bands of products obtained by the two-time amplification of 3-valent rotavirus gene reassortment seedlings.

From the above electrophoresis results, no specific expected band was amplified in the detected rotavirus vaccine and related components.

8.2 sequencing results and comparison

Respectively sending DNA fragments obtained by 2 times of PCR amplification of two types of universal primers and various types of specific primers to the Shanghai workers for sequence determination, wherein PCV-2 cell culture is taken as a template, and 2 nd PCR products of the universal primers and the 2 nd specific primers are directly and respectively sequenced by respective sense primers; and (3) taking the PCV-1 positive recombinant plasmid as a template, constructing a2 nd PCR product of a universal primer and a type 1 specific primer into a T vector, and sequencing.

Wherein, the DNA fragment sequence sequencing result obtained by 2 times of PCR amplification of the two types of universal primers is shown as SEQ NO. 13 (corresponding to PCV-I,260bp) and is shown as SEQ NO. 14 (corresponding to PCV-2, 240bp), and the DNA fragment sequence sequencing result obtained by 2 times of PCR amplification of the PCV-I type specific primers is shown as SEQ NO. 15 (434 bp); the sequencing result of the DNA fragment obtained by 2 times of PCR amplification of the PCV-II type specific primer is shown as SEQ NO:16 (479 bp).

The nucleotide sequences determined above were aligned in NCBI BLAST and the results were: the nucleotide sequence of the 260bp fragment (the product of PCV-2 porcine kidney cell culture amplified by the type 2 universal primer) has 97 percent of sequence homology with PCV-II, the nucleotide sequence of the 260bp fragment (the PCV-1 recombinant plasmid amplified by the type 2 universal primer) has 100 percent of homology with the synthesized PCV1 fragment, and the nucleotide homology with other PCV1 is up to more than 96 percent; the homology of the nucleotide of the 434bp fragment (PCV-I type specific primer amplification product) and the synthesized PCV1 fragment is 100 percent, and the nucleotide homology with other PCV1 is also up to more than 99 percent; the nucleotide sequence of the 500bp fragment (PCV-II type specific primer amplification product) has more than 98 percent of homology with the fragment of PCV2, and the homology is shown in the following tables 2 to 5.

Table 2 shows the nucleotide sequence comparison of fragments obtained by amplifying PCV2 cell culture with universal primers

Numbering	Description	Homology of
			JQ002672.1	Porcine circovirus 2 strain Bj2010PG,complete genome	97％
JN388690.1	Porcine circovirus 2 isolate PCV2-LJR,complete genome	97％
			JF317579.1	Porcine circovirus 2 isolate A4275,complete genome	97％
FJ804417.1	Porcine circovirus 2 isolate PCV2-Ha08,complete genome	97％
			GU325754.1	Porcine circovirus 2 isolate SY4,complete genome	97％
GQ227412.1	Porcine circovirus 2 isolate JSDT0706,complete genome	97％
			FJ905468.1	Porcine circovirus 2 isolate P710-1,complete genome	97％
EU257513.1	Porcine circovirus 2 strain ZJU0601,complete genome	97％
			EF493852.1	Porcine circovirus 2 strain ZZ rep gene,complete cds	97％

Table 3 shows the nucleotide sequence comparison results of the fragments obtained by amplifying PCV1 recombinant plasmid with universal primers

Numbering	Description	Homology of
			GU799575.1	Porcine circovirus 1 strain NMB,complete genome	100％
GQ449671.1	Porcine circovirus 1 strain HRB-09,complete genome	100％
			DQ358813.1	Porcine circovirus 1 strain SC-1,complete genome	100％
AY099501.1	Porcine circovirus 1 isolate CT-PCV-P7,complete genome	100％
			Y09921.1	Porcine circovirus 1,complete genome	100％
GU371908.1	Porcine circovirus 1 isolate Tian Jin,complete genome	99％
			EF533941.1	Porcine circovirus 1 strain HZ2006,complete genome	99％
AF071879.1	Porcine circovirus 1,complete genome	99％
			DQ472013.1	Porcine circovirus 1 strain sc-3,complete genome	99％
GQ404925.1	Porcine circovirus 1 isolate Pork991	98％
			KC894933.1	Porcine circovirus 1 strain GY,complete genome	96％

Table 4 shows the results of comparing the nucleotide sequences of the amplified fragments of the type I specific primers

Numbering	Description	Homology of
			KJ408799.1	Porcine circovirus 1 isolate PCV1-Hun,complete genome	100％
JX566507.1	Porcine circovirus 1 strain NJ03,complete genome	100％
			GU799575.1	Porcine circovirus 1 strain NMB,complete genome	100％
EF493843.1	Porcine circovirus 1 strain SC-11,complete genome	100％
			DQ650650.1	Porcine circovirus 1 strain PK,complete genome	100％
U49186.1	Porcine circovirus 1,complete genome	100％
			KC447455.1	Porcine circovirus 1 isolate PCV1-XFD-Beijing,complete genome	99％
DQ648032.1	Porcine circovirus 1 isolate WB-H-8,complete genome	99％

Table 5 shows the results of comparing the nucleotide sequences of the amplified fragments of type II specific primers

It should be noted that the embodiments of the present invention have been described in terms of preferred embodiments, and not by way of limitation, and that those skilled in the art can make modifications and variations of the embodiments described above without departing from the spirit of the invention.

Claims

1. A method for detecting porcine circovirus for non-disease diagnostic and therapeutic purposes, wherein the porcine circovirus includes both type I and type II viruses, the method comprising:

extracting DNA from a sample, and carrying out first PCR; and

performing a second PCR on the amplification product obtained by the first PCR,

the primers used in the first PCR comprise a first primer group, a second primer group and a third primer group,

the primers used in the second PCR comprise a fourth primer group, a fifth primer group and a sixth primer group,

wherein,

2. The detection method according to claim 1, wherein the sample is rotavirus oral live vaccine, 3-valent rotavirus reassortant vaccine, calf serum, a culture medium, pancreatin for cell digestion, pancreatin for virus digestion, rotavirus LD strain, rotavirus LS strain, rotavirus LH strain and/or Vero cell.

3. The detection method of claim 1, wherein the method further comprises performing a qualitative analysis of the product after the second PCR amplification.

4. The detection method of claim 3, wherein the qualitative analysis comprises visualizing or sequencing the products after the second PCR amplification using gel electrophoresis.

5. A primer group for detecting porcine circovirus, wherein the porcine circovirus comprises type I and/or type II viruses, and is characterized in that the primer group comprises an upstream primer and a downstream primer, wherein the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 1 and a downstream primer with a sequence shown as SEQ NO. 2.

6. A primer group for detecting porcine circovirus, wherein the porcine circovirus comprises type I and/or type II viruses, and is characterized in that the primer group comprises an upstream primer and a downstream primer, wherein the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 1 and a downstream primer with a sequence shown as SEQ NO. 7.

7. A primer group for detecting porcine circovirus, wherein the porcine circovirus comprises type I virus, and is characterized in that the primer group comprises an upstream primer and a downstream primer, wherein the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 5 and a downstream primer with a sequence shown as SEQ NO. 6.

8. A primer group for detecting porcine circovirus, wherein the porcine circovirus comprises type I virus, and is characterized in that the primer group comprises an upstream primer and a downstream primer, wherein the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 10 and a downstream primer with a sequence shown as SEQ NO. 11.

9. A primer group for detecting porcine circovirus, wherein the porcine circovirus comprises II type viruses, and is characterized in that the primer group comprises an upstream primer and a downstream primer, wherein the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 3 and a downstream primer with a sequence shown as SEQ NO. 4.

10. A primer group for detecting porcine circovirus, wherein the porcine circovirus comprises II type viruses, and is characterized in that the primer group comprises an upstream primer and a downstream primer, wherein the upstream primer comprises an upstream primer with a sequence shown as SEQ NO. 8 and a downstream primer with a sequence shown as SEQ NO. 9.