CN116004922B - Dual fluorescence PCR kit for detecting PCV2 and PCV3 of pigs - Google Patents
Dual fluorescence PCR kit for detecting PCV2 and PCV3 of pigs Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a dual fluorescence PCR kit and a detection method for detecting PCV2 and PCV3 of pigs. The dual fluorescent PCR kit for detecting the PCV2 and PCV3 of the invention comprises PCV2 primer, probe sequence, PCV3 primer and probe sequence, negative control, positive control, 2X fluorescent PCR reaction solution and ddH 2 O. The invention designs primers and probes for detecting PCV2 and PCV3 by referring to the clinically detected PCV2 and PCV3 strains, and the primers and probes are assembled into a kit, so that almost all known PCV2 and PCV3 strains can be amplified. The dual fluorescence PCR kit has the characteristics of high sensitivity, high specificity, good repeatability and the like, and has important significance for clinically performing differential diagnosis of PCV2 and PCV3.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a dual fluorescence PCR kit and a detection method for detecting PCV2 and PCV3 of pigs.
Background
Porcine circovirus (Porcine circovirus, abbreviated: PCV) is one of the smallest animal viruses discovered so far. Three serotypes of PCV are known, namely PCV1, PCV2 and PCV3.PCV1 is a non-pathogenic virus. PCV2 is a pathogenic virus, which is the main pathogen of the multisystemic wasting syndrome (Postweaning Multisystemic Wasting Syndrome, PMWS) in weaned pigs. The disease was first found in Canada in 1991 and rapidly developed and prevailed in Europe and America, including in Asia in China. In addition to PMWS, diseases such as PDNS (swine dermatitis and nephrotic syndrome), PNP (necrotizing PNP), PRDC (porcine respiratory disease syndrome), reproductive disorders, congenital tremors, enteritis, etc. are also associated with PCV2 infection. PCV2 and related swine diseases thereof have different mortality rates of 10% -30%, and the death rate of a severe swine farm is up to 40% when the disease is outbreak, so that serious economic loss is caused to the pig industry. PMWS has been recognized by veterinarians and pig breeders worldwide as an important infectious disease that is newly discovered to cause swine immune dysfunction following Porcine Reproductive and Respiratory Syndrome (PRRS).
A novel circovirus, porcine circovirus type 3 (PCV 3), was isolated from sows in the United states with acute death in 2016 and associated clinical symptoms like swine dermatitis and nephrotic syndrome and showing reproductive dysfunction. Studies have shown that PCV3 may play a pathogenic role in reproductive disorders and swine dermatitis and nephrotic syndrome. The PCV3 infection is widely distributed in various tissues and organs, and the tissue and organs with more common case change are heart and lung injuries. PCV3 can cause clinical manifestations like PCV2 infection onset, such as dermatitis nephrotic syndrome and sow reproductive dysfunction, PCV2 vaccine immunization is strictly done clinically, and PCV3 detection and monitoring are enhanced.
PCV3 is a novel porcine circovirus which has a far-reaching genetic evolution relationship with PCV2 and a large difference, and PCV2 vaccine cannot provide protection for PCV3 in clinic. Therefore, it is very important to differential diagnosis of PCV2 and PCV3.
At present, the PCV2 is detected by national standards GB/T34745-2017 and GB/T35901-2018, but through analysis, many dominant PCV2 virus strains cannot be detected by using primers and probes of GB/T34745-2017 and GB/T35901-2018. Such as: neither GB/T34745-2017 nor GB/T35901-2018 can amplify the PCV2 sequence NCBI number MK 015043. Likewise, many NCBI No. (FJ 644927 and MW051676, etc.) PCV2 strains could not be detected using PCV2 detection reagents of the prior published patent (patent No. CN 202111401011). Similar problems as described above exist with patent number CN 201010271159.9.
Disclosure of Invention
The invention aims to provide a double fluorescence PCR kit for detecting PCV2 and PCV3 of pigs.
According to the dual fluorescent PCR kit for detecting the PCV2 and PCV3 of the specific embodiment of the invention, the kit comprises PCV2 primers, a probe sequence and PCV3 primers and a probe sequence, wherein,
PCV2 primer sequence:
SEQ ID NO.1: 5’- TCCTCCCGCCATACCATAAC -3’；
SEQ ID NO.2: 5’- CGAACGCAGTGCCGAGGCCTAC -3’；
PCV2 probe sequence:
SEQ ID NO.3: 5’- CAGCCCTTCTCCTACCACTCCCGSTA -3’；
PCV3 primer sequence:
SEQ ID NO.5：5’- AGACCCCGCCCAAGGA -3’；
SEQ ID NO.6：5’- GAAGTGGTTGGCGTGCCAGGGCTT -3’；
PCV3 probe sequences
SEQ ID NO.7：5’- ACGACGCCACAGAAGGCGCTATGYC -3’。
The 5 'end of the probe is marked with a fluorescent group, the 3' end is marked with a quenching group, wherein the fluorescent group can be FAM, HEX, JOE, TET, CY3, CY5, ROX, texas and the like, and the preferable scheme of the invention is FAM and ROX; the quenching groups can be TAMRA, BHQ (BHQ 1, BHQ2, BHQ 3), MGB and the like, and the preferable scheme of the invention is BHQ1 and BHQ2.
Specifically, the PCV2 probe sequence is:
Fam-5’- CAGCCCTTCTCCTACCACTCCCGSTA -3’-BHQ1；
PCV3 probe sequence: ROX-5 '-ACGACGCCACAGAAGGCGCTATGYC-3' -BHQ2.
According to the dual fluorescence PCR kit for detecting the PCV2 and PCV3 of the pigs, the concentration ratio of the primer to the probe is 1-5:1, a step of; wherein, the primer concentration is 100-200nM, and the probe concentration is 20-200 nM.
According to the dual fluorescent PCR kit for detecting the PCV2 and PCV3 of the pig, which is provided by the specific embodiment of the invention, the kit further comprises a negative control, a positive control, a 2X fluorescent PCR reaction solution and ddH 2 O。
Wherein the negative control is DNase-free ddH 2 O。
According to the dual fluorescent PCR kit for detecting the PCV2 and PCV3 of the specific embodiment of the invention, the positive control is plasmid or pseudovirus, and the positive control comprises PCV2 amplified product sequence and PCV3 amplified product sequence.
Wherein, the PCV2 positive control sequence is shown in SEQ ID NO. 4:
TCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTCCCGCTACTTTACCCCCAAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGGCTACAAACTGCTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCG；
the PCV3 positive control sequence is shown in SEQ ID NO. 8:
ATGAGACACAGAGCTATATTCAGAAGAAGACCCCGCCCAAGGAGGCGACGACGCCACAGAAGGCGCTATGTCAGAAGAAAACTATTCATTAGGAGGCCCACAGCTGGCACATACTACACAAAGAAATACTCCACCATGAACGTCATTTCCGTTGGAACCCCTCAGAATAACAAGCCCTGGCACGCCAACCACTTCATTACCCGCCTAAACGAATGGGAAAC。
according to the dual fluorescent PCR kit for detecting the PCV2 and PCV3 of the pig, the plasmid is pUC57, and one of the pseudovirus type MS2 phage or recombinant adenovirus is adopted.
According to the dual fluorescence PCR kit for detecting the PCV2 and PCV3 of the pigs, the concentration of the plasmid is 1.0x10 8 -1.0x10 9 copy/ml, the pseudovirus concentration is 1.0x10 8 -1.0x10 9 copy/ml。
The invention also provides a use method of the double fluorescence PCR kit for detecting the PCV2 and PCV3 of the pigs,
(1) The reaction system is 20-50 mu L, and each reaction well comprises 2 Xfluorescent PCR reaction liquid, a sample to be detected (negative control or positive control) and ddH 2 O, etc.;
(2) The reaction conditions are as follows: 50 ℃ for 3min; pre-denaturation at 95℃for 2min, denaturation at 95℃for 15s, annealing at 60℃for 30s, and fluorescence collection for 40-45 cycles.
(3) Analysis of results:
the test establishment condition is as follows: negative control wells, FAM, ROX channels all had no Ct value; the CT value of the positive control hole FAM and ROX channel is less than or equal to 30.00.
And (3) result judgment: if the FAM (ROX) Ct value is less than or equal to 38.00, the FAM (ROX) Ct value is 38.00< if the FAM (ROX) Ct value is less than 40.00, the FAM (ROX) Ct value is suspicious, and otherwise, the FAM (ROX) Ct value is negative; if FAM positive indicates porcine PCV2 pathogen, if ROX positive indicates porcine PCV3 pathogen.
The sample to be tested for which the kit of the invention is applicable comprises an environmental sample, an anus swab, viscera tissue or blood of pigs, and the like.
The invention has the beneficial effects that:
the invention designs the primers and probes for PCV2 and PCV3, and the primers and probes are assembled into the kit, so that the covered PCV2 and PCV3 strains are detected in a wide range, and meanwhile, the linear relation of the amplification curve is good, the variation coefficient is small, and the sensitivity, the specificity and the repeatability are high.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a concentration of 10 in example 3 8 、10 7 、10 6 、10 5 、10 4 And 10 3 PCV2 fluorescent quantitative PCR amplification curve of copy/. Mu.l positive plasmid;
FIG. 2 is a standard equation based on the amplification curve of FIG. 1, with the abscissa being the template concentration (to the power N of 10) and the ordinate corresponding to the Ct value;
FIG. 3 shows a concentration of 10 in example 3 8 、10 7 、10 6 、10 5 、10 4 And 10 3 PCV3 fluorescent quantitative PCR amplification curve of copy/. Mu.l positive plasmid;
FIG. 4 is a standard equation based on the amplification curve of FIG. 3, with the abscissa being the template concentration (to the power N of 10) and the ordinate corresponding to the Ct value.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Example 1 primers, probes and positive controls
Many variant strains of PCV2 exist, with the strain sequences recorded in NCBI including, but not limited to, the following:
FJ644929，FJ644559，MK015043，KT719404，MW051676，OM274023.1，OM274022.1，OM274020.1，OM274019.1，OM274018.1，OM274017.1，OM858856.1，OM858855.1，OM858854.1，OP413469.1，OP413468.1，OP413467.1，OP413466.1，MW369443.1，OM743483.1，OM743482.1，OM743481.1，OM743479.1，OM743477.1，OM743476.1，OM743472.1，OM743468.1，OM743464.1，OM743461.1，OM743460.1，OM743459.1，OM743457.1，OM743456.1，OM681503.1，OM681502.1，OM681500.1，OM681497.1，OM681490.1，OM681488.1，OM681486.1，OM681482.1，OM681481.1，OM681480.1，OM6 81479.1，OM681477.1，OM681476.1，OM681475.1，OM681471.1，OM681470.1，OM681468.1，OM681466.1，OM681465.1，OM681463.1，OM681462.1，OM681459.1，OM681458.1，OM681457.1，OM681452.1，OM681451.1，OM677751.1，OM677749.1，OM677748.1，OM677747.1，OM677746.1，OM677744.1，OM677743.1，OM677742.1，OM677741.1，OM677738.1，OM677737.1，OM677736.1，OM677735.1，OM677734.1，OM677733.1，OM677732.1，OM677731.1，OM677730.1，OM677729.1，OM677728.1，OM677727.1，OM677726.1，OM677724.1，OM677723.1，OM677721.1，OM677720.1，OM677719.1，OM677717.1，OM677716.1，OM677714.1，OM677713.1，OM677712.1，OM677711.1，OM677710.1，OP441380.1，OM460170.1，OM288665.1，OM288664.1，OM288663.1，OM288662.1，OM288661.1，OM288660.1，OM288659.1，OM288658.1。
many variant strains of PCV3 also exist, wherein the strain sequences recorded in NCBI include, but are not limited to, the following sequences:
ON184737.1，MZ004438.1，OX326964.1，ON184728.1，ON184729.1，ON184730.1，ON184731.1，ON184732.1，ON184733.1，ON184734.1，ON184735.1，ON184736.1，ON184738.1，ON184739.1，ON184740.1，ON184743.1，ON184747.1，ON184748.1，ON184749.1，ON184750.1，ON184751.1，MZ747123.1，MZ747124.1，MZ747125.1，MZ667331.1，MZ667330.1，MZ667332.1，MZ667333.1，MZ667334.1，OL799306.1，OL619331.1，OL619332.1，OL619335.1，OL619336.1，OL619337.1，OL619338.1，OL619339.1，OL619339.1，OL619339.1，OL619339.1，OL619339.1，OL619339.1，OL619339.1，OM249966.1，OM032567.1，OM032566.1，OL956951.1，OL956950.1，OP066329.1，OP066328.1，OP066327.1，OP066326.1，OP066325.1，OP066324.1，OP066323.1，OP066322.1，OP066321.1，OP066320.1，OP066319.1，OP066318.1，OP066317.1，OP066316.1，OP066315.1，OP066314.1，ON989005.1，ON586851.1，CP110845.1，CP110836.1，CP090311.1，OL619374.1，OL619373.1，OL619372.1，OL619371.1，OL619370.1，OL619369.1，OL619368.1，OL619366.1，OL619365.1，OL619364.1，OL619363.1，OL619362.1，OL619361.1，OL619360.1，OL619359.1，OL619358.1，OL619357.1，OL619356.1，OL619355.1，OL619354.1，OL619352.1，OL619351.1，OL619348.1，OL619347.1，OL619346.1，OL619345.1，OL619344.1，OL619343.1，OL619342.1，OL619341.1，OL619340.1。
the inventor designs a plurality of groups of primers for PCV2 and PCV3 according to the sequences, and only the following primers and probes can be amplified to obtain the strain sequences through experimental screening,
PCV2 primer and probe sequences were as follows:
PCV2-ORF2-175F: 5’-TCCTCCCGCCATACCATAAC-3’；
PCV2-ORF2-175R: 5’-cgaacgcagtgccgaggcctac-3’；
PCV2-ORF2-175P: Fam-5’-CAGCCCTTCTCCTACCACTCCCGSTA-3’-BHQ1；
PCV3 primer and probe sequences were as follows:
PCV3-ORF2-168F：5’-AGACCCCGCCCAAGGA-3’；
PCV3-ORF2-168R：5’-Gaagtggttggcgtgccagggctt-3’；
PCV3-ORF2-168P：ROX-5’-ACGACGCCACAGAAGGCGCTATGYC-3’-BHQ2。
meanwhile, the invention selects SEQ ID NO.4 sequence, and clones the SEQ ID NO.4 sequence on a pUC57 vector to assemble positive control pUC57-SEQ NO.4 of PCV 2. Similarly, the invention selects SEQ ID NO.8 sequence, and clones the SEQ ID NO.8 sequence on a pUC57 vector to assemble positive control pUC57-SEQ NO.8 of PCV 3;
SEQ ID NO.4：
TCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTCCCGCTACTTTACCCCCAAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGGCTACAAACTGCTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCG；
SEQ ID NO.8：
ATGAGACACAGAGCTATATTCAGAAGAAGACCCCGCCCAAGGAGGCGACGACGCCACAGAAGGCGCTATGTCAGAAGAAAACTATTCATTAGGAGGCCCACAGCTGGCACATACTACACAAAGAAATACTCCACCATGAACGTCATTTCCGTTGGAACCCCTCAGAATAACAAGCCCTGGCACGCCAACCACTTCATTACCCGCCTAAACGAATGGGAAAC
example 2 Dual fluorescence PCR kit for detecting porcine PCV2 and PCV3
1. 2x PCR reaction solution; purchasing self-made holothurian is a gene technology company;
2. the primers and probes were diluted with DEPC water to a concentration of 10. Mu.M.
PCV2 primer probe sequence:
PCV2-ORF2-175F: 5’-TCCTCCCGCCATACCATAAC-3’；
PCV2-ORF2-175R: 5’-CGAACGCAGTGCCGAGGCCTAC-3’；
PCV2-ORF2-175P: Fam-5’-CAGCCCTTCTCCTACCACTCCCGSTA-3’-BHQ1；
PCV3 primer probe sequence:
PCV3-ORF2-168F：5’-AGACCCCGCCCAAGGA-3’；
PCV3-ORF2-168R：5’-GAAGTGGTTGGCGTGCCAGGGCTT-3’；
PCV3-ORF2-168P：ROX-5’-ACGACGCCACAGAAGGCGCTATGYC-3’-BHQ2；
3. positive control dilution:
PCV2 positive control pUC57-SEQ NO.4 diluted to 1.0x10 8 -1.0x10 9 Second-order copy/ml.
PCV3 positive control pUC57-SEQ NO.8 diluted to 1.0x10 8 -1.0x10 9 Second-order copy/ml.
4. Kit assembly (50T):
(1) One fluorescence PCR reaction MIX (900 μl): comprises the following components
(2) A negative control (0.5 ml: ddH) 2 O）
(3) A positive control (0.5 ml: pUC57-SEQ NO.4, pUC57-SEQ NO. 8).
(4) One ddH 2 O（1ml）。
(5) Instructions for use 1 part.
4. Determination criteria of detection results:
1) And (3) quality control: negative control: FAM channel detection has no Ct value, ROX channel detection has no Ct value;
positive control: the detection Ct value of the FAM channel is less than or equal to 30.00, and the detection Ct value of the ROX channel is less than or equal to 30.00;
the negative and positive control detection results are required to be established simultaneously, and the detection results are effective.
2) And (3) result judgment:
the following can be directly determined:
remarks: if the Ct value of 38.00< is detected to be less than 40.00, the rechecking is needed, and if the rechecking has the Ct value, the rechecking is judged to be positive; if there is no ct value, it is determined as negative.
Example 3 verification of sensitivity, specificity and repeatability of Dual fluorescence PCR kit for porcine PCV2 and PCV3
1. Genomic DNA extraction of samples:
the method for extracting genomic DNA such as environmental sample, anus swab, and viscera tissue or blood of pig can be manually extracted, or can be used in kit or nucleic acid extractor.
2. Linearity, sensitivity
Each positive sample was subjected to 10-fold gradient dilution of positive control plasmid as a template, i.e., 10 8 、10 7 、10 6 、10 5 、10 4 And 10 3 copy/. Mu.l positive plasmid was used as template and the test of this example was performed according to the method of example 2, the results are shown in Table 1:
the detection result of PCV2 is shown in FIG. 1 and FIG. 2; results of PCV3 detection FIGS. 3 and 4.
The results show that the detection range of the method is 10 8 -10 3 The virus content in this range gives accurate results, i.e.the sensitivity of the method allows detection of samples with a virus content of a minimum of 10 copies.
3. Specificity (specificity)
In order to detect the specificity of the kit of the present invention, samples such as Streptococcus suis, actinobacillus, haemophilus parasuis, pseudorabies virus, african swine fever virus, parvovirus, and encephalitis B virus were detected using the kit of the present invention according to the method of example 2, and the results are shown in Table 2:
the result shows that the detection of the viruses can obtain negative results, and the specificity of the kit is good.
4. Repeatability test
In order to detect the reproducibility of the kit of the present invention, the concentration of 10 was detected using the kit of the present invention 6 And 10 5 Positive control of (c) in (a). The results are shown in Table 3:
as can be seen from the above table, the coefficient of variation is small, and the repeatability is good.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. A dual fluorescent PCR kit for detecting PCV2 and PCV3 of pigs is characterized by comprising PCV2 primers, a probe sequence, PCV3 primers and a probe sequence,
PCV2 primer sequence:
SEQ ID NO.1: 5’-TCCTCCCGCCATACCATAAC-3’；
SEQ ID NO.2: 5’-CGAACGCAGTGCCGAGGCCTAC-3’；
PCV2 probe sequence:
SEQ ID NO.3: Fam-5’-CAGCCCTTCTCCTACCACTCCCGSTA-3’-BHQ1；
PCV3 primer sequence:
SEQ ID NO.5：5’-AGACCCCGCCCAAGGA-3’；
SEQ ID NO.6：5’-GAAGTGGTTGGCGTGCCAGGGCTT-3’；
PCV3 probe sequence:
SEQ ID NO.7：ROX-5’-ACGACGCCACAGAAGGCGCTATGYC-3’-BHQ2；
the concentration ratio of the primer to the probe is 1-5:1; wherein, the primer concentration is 100-200nM, and the probe concentration is 20-200nM;
the kit also comprises a negative control, a positive control, 2X fluorescence PCR reaction liquid and ddH 2 O;
The negative control is ddH without DNase 2 O;
The positive control is pUC57 plasmid, and the positive control contains PCV2 amplified product sequence and PCV3 amplified product sequence;
the PCV2 amplification product sequence is:
SEQ ID NO.4:
TCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTCCCGCTACTTTACCCCCAAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGGCTACAAACTGCTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCG;
the PCV3 amplification product sequence is:
SEQ ID NO.8:
ATGAGACACAGAGCTATATTCAGAAGAAGACCCCGCCCAAGGAGGCGACGACGCCACAGAAGGCGCTATGTCAGAAGAAAACTATTCATTAGGAGGCCCACAGCTGGCACATACTACACAAAGAAATACTCCACCATGAACGTCATTTCCGTTGGAACCCCTCAGAATAACAAGCCCTGGCACGCCAACCACTTCATTACCCGCCTAAACGAATGGGAAAC;
plasmid concentration was 1.0X10 8 -1.0×10 9 copy/ml。
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