CN107475451B - European and American dual microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome virus - Google Patents
European and American dual microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome virus Download PDFInfo
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Abstract
The invention discloses a European and American dual microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome virus. The primer probe combination provided by the invention consists of LV-F1R1P1 and VR2332-F1R1P 1; LV-F1R1P1 is composed of LV-F1, LV-R1 and LV-P1; LV-F1 is shown in a sequence 1; LV-R1 is shown as a sequence 2; LV-P1 is shown as sequence 3; VR2332-F1R1P1 consists of VR2332-F1, VR2332-R1 and VR 2332-P1; VR2332-F1 is shown as sequence 4; VR2332-R1 is shown as sequence 5; VR2332-P1 is shown in sequence 6. The invention has great application value for the prevention and control of European type and/or American type of the porcine reproductive and respiratory syndrome virus, and is beneficial to controlling epidemic situation from the source and effectively preventing large-scale outbreak of the porcine epidemic disease.
Description
Technical Field
The invention belongs to the field of virus detection, and particularly relates to a European and American dual microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome viruses.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious infectious disease characterized by reproductive disorders in pregnant sows, particularly dyspnea in suckling piglets, caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The disease first outbreaks in the united states in 1987, followed by successive appearance in canada, europe and asia, has now become prevalent worldwide, causing enormous economic losses each year. Our country first developed PRRS in 1995 and then rapidly spread. In 2006, a high fever of a pig with high body temperature, high morbidity and high mortality as main clinical characteristics is completely developed in China, and the pathogen of the disease is a highly pathogenic porcine reproductive and respiratory syndrome virus which is mutated by classical PRRSV (porcine reproductive and respiratory syndrome) which is popular in China before. To date, PRRSV has two types, namely the american type represented by atcvr-2332 (VR strain) strain and the european type represented by Lelystadvirus (LV strain), and the american type is divided into two subtypes, a classical american strain and a highly pathogenic american strain, and the three subtypes have some differences in gene sequence. The main epidemic in China is the American strain, and the European strain is occasionally found in China, so that a detection method capable of quickly diagnosing PRRSV is clinically needed.
The traditional PRRSV detection method mainly comprises the separation and identification of virus, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA), indirect enzyme-linked immunosorbent assay (indirect ELISA) and the like. The most commonly used methods for nucleic acid detection at present are RT-PCR and fluorescent RT-PCR. The traditional PRRSV detection method has the defects of high-cost instrument and equipment, long time required by the test and the like. Although RT-PCR and fluorescence RT-PCR have the characteristics of stronger specificity, high sensitivity, good repeatability, high automation degree and the like compared with the prior method, the methods can only realize qualitative and semi-quantitative detection, cannot accurately quantify the virus nucleic acid, and still have certain limitations on sensitivity and sensitivity specificity.
The concept of Digital PCR (dPCR) was adopted by Bert Vogelstein in 1999 as early as the associated document of concurrence, which was originally intended to be able to detect minute amounts of mutant cells from a large number of normal somatic cells in clinical samples (e.g. urine, lymph fluid, plasma, feces, etc.), but it has not been possible to embody the core concept of Digital PCR very well-the "infinite dilution" (tertiary dilution) because the consumable used to dilute the samples at that time was only a 384-well plate. The technology of microdroplet at the core of QX200 system of Bio-Rad company can divide a sample into microdroplets with 20,000 nano-upgrades, essentially changes one test of traditional quantitative PCR into 20,000 tests, greatly improves the sensitivity and accuracy of nucleic acid sequence detection, is a perfect deduction of the concept of 'infinite dilution', and the principle of the method can be called as droplet digital PCR (ddPCR). The QX200ddPCR system includes two instruments: droplet generators and droplet analyzers, and their associated consumables. The droplet generator divides each sample into 20,000 uniform nanoliter droplets, each of which either contains no nucleic acid target molecules to be detected or contains one to several nucleic acid target molecules to be detected. Each droplet acts as a separate PCR reactor. The droplets were then transferred to a 96-well PCR plate for end-point PCR amplification. And (3) detecting each droplet one by adopting a droplet analyzer (droplet reader), judging the droplet with a fluorescent signal as 1, judging the droplet without the fluorescent signal as 0, and finally calculating the concentration or copy number of the target molecule to be detected by analysis software according to a Poisson distribution principle and the proportion of positive droplets. Compared with the traditional quantitative PCR, the accuracy and sensitivity of the digital PCR are better. By using the droplet-type digital PCR technology, researchers can detect rare mutations, accurately determine copy number variation, and perform absolute quantification on gene expression. By virtue of the high sensitivity of the QX200 system, researchers can now detect target molecules at concentrations as low as 1/1,000,000.
Disclosure of Invention
The invention aims to provide a European and American double microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome virus.
The invention provides a primer probe combination, which consists of a primer probe group LV-F1R1P1 and a primer probe group VR2332-F1R1P 1;
the primer probe group LV-F1R1P1 consists of a primer LV-F1, a primer LV-R1 and a probe LV-P1;
the primer LV-F1 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the primer LV-R1 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the probe LV-P1 has a fluorescence reporter group at one end and a fluorescence quenching group at the other end, and the nucleotide sequence is (a5) or (a 6):
(a5) as shown in sequence 3 of the sequence table;
(a6) as shown by the substitution and/or deletion and/or addition of one or more nucleotides to the sequence 3;
the primer probe group VR2332-F1R1P1 consists of a primer VR2332-F1, a primer VR2332-R1 and a probe VR 2332-P1;
the primer VR2332-F1 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer VR2332-R1 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
(a4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;
the probe VR2332-P1 has a fluorescence reporter group at one end and a fluorescence quencher group at the other end, and has the following nucleotide sequence (a5) or (a 6):
(a5) as shown in sequence 6 of the sequence table;
(a6) as shown by the substitution and/or deletion and/or addition of one or more nucleotides to the sequence 6;
the probe LV-P1 and the probe VR2332-P1 have fluorescent reporter groups of different colors.
Specifically, the probe LV-P1 was labeled with a fluorophore FAM at the 5 'end and a fluorescence quencher BHQ1 at the 3' end.
Specifically, the 5 'end of the probe VR2332-P1 is labeled with a fluorescent group HEX, and the 3' end is labeled with a fluorescence quenching group BHQ 1.
The application of the primer probe combination is (b1), (b2), (b3), (b4), (b5) or (b 6):
(b1) identifying a porcine reproductive and respiratory syndrome virus european type and/or a porcine reproductive and respiratory syndrome virus american type;
(b2) preparing a kit for identifying porcine reproductive and respiratory syndrome virus European type and/or porcine reproductive and respiratory syndrome virus American type;
(b3) detecting whether the sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus;
(b4) preparing a kit for detecting whether a sample to be detected contains the European type of porcine reproductive and respiratory syndrome virus and/or the American type of porcine reproductive and respiratory syndrome virus;
(b5) detecting the content of European type and/or American type of the porcine reproductive and respiratory syndrome virus in a sample to be detected;
(b6) preparing a kit for detecting the European type and/or American type content of the porcine reproductive and respiratory syndrome virus in a sample to be detected.
The invention also protects the application of the primer probe combination, which is (b1), (b2), (b3), (b4), (b5) or (b 6):
(b1) identifying a porcine reproductive and respiratory syndrome virus european type and/or a porcine reproductive and respiratory syndrome virus american type;
(b2) preparing a kit for identifying porcine reproductive and respiratory syndrome virus European type and/or porcine reproductive and respiratory syndrome virus American type;
(b3) detecting whether the sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus;
(b4) preparing a kit for detecting whether a sample to be detected contains the European type of porcine reproductive and respiratory syndrome virus and/or the American type of porcine reproductive and respiratory syndrome virus;
(b5) detecting the content of European type and/or American type of the porcine reproductive and respiratory syndrome virus in a sample to be detected;
(b6) preparing a kit for detecting the European type and/or American type content of the porcine reproductive and respiratory syndrome virus in a sample to be detected.
The invention also provides a kit, which comprises the primer probe combination; the function of the kit is as follows (c1), (c2) or (c 3): (c1) identifying a porcine reproductive and respiratory syndrome virus european type and/or a porcine reproductive and respiratory syndrome virus american type; (c2) detecting whether the sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus; (c3) and detecting the content of the European type and/or the American type of the porcine reproductive and respiratory syndrome virus in the sample to be detected.
The kit further comprises a carrier carrying method I and/or method II and/or method III.
The invention also provides a method (method I) for detecting whether the virus to be detected is the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus, which comprises the following steps: taking total RNA of the virus to be detected as a template, and adopting the primer probe combination to perform digital RT-PCR; if the detection result based on the fluorescent group corresponding to the probe LV-P1 is positive, the virus to be detected is or is selected as the European porcine reproductive and respiratory syndrome virus, if the detection result based on the fluorescent group corresponding to the probe LV-P1 is negative, the virus to be detected is or is selected as the European non-porcine reproductive and respiratory syndrome virus; if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is positive, the virus to be detected is or is candidate to be the porcine reproductive and respiratory syndrome virus American type, and if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is negative, the virus to be detected is or is candidate to be the non-porcine reproductive and respiratory syndrome virus American type.
The invention also provides a method (method II) for detecting whether a sample to be detected contains the European type of porcine reproductive and respiratory syndrome virus and/or the American type of porcine reproductive and respiratory syndrome virus, which comprises the following steps: taking total RNA of a sample to be detected as a template, and adopting the primer probe combination to perform digital RT-PCR; if the detection result based on the fluorescent group corresponding to the probe LV-P1 is positive and the sample to be detected contains the European porcine reproductive and respiratory syndrome virus, if the detection result based on the fluorescent group corresponding to the probe LV-P1 is negative and the sample to be detected does not contain the European porcine reproductive and respiratory syndrome virus; if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is positive, the sample to be detected contains the porcine reproductive and respiratory syndrome virus American type, and if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is negative, the sample to be detected does not contain the porcine reproductive and respiratory syndrome virus American type.
The invention also provides a method (method III) for detecting the content of the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus in a sample to be detected, which comprises the following steps: taking total RNA of a sample to be detected as a template, and adopting the primer probe combination to perform digital RT-PCR; obtaining the European content of the porcine reproductive and respiratory syndrome virus in a sample to be detected according to the number of the European positive droplets of the porcine reproductive and respiratory syndrome virus, wherein the European positive droplets of the porcine reproductive and respiratory syndrome virus are droplets with positive detection results based on fluorescent groups corresponding to a probe LV-P1; and obtaining the American type content of the porcine reproductive and respiratory syndrome virus in the sample to be detected according to the number of the American type positive droplets of the porcine reproductive and respiratory syndrome virus, wherein the American type positive droplets of the porcine reproductive and respiratory syndrome virus are droplets with positive detection results based on fluorescent groups corresponding to the probes VR 2332-P1.
In the method I or the method II or the method III, in the reaction system of the digital RT-PCR, the concentration of a primer LV-F1 is 0.9 mu M, the concentration of a primer LV-R1 is 0.9 mu M, the concentration of a probe LV-P1 is 0.2 mu M, the concentration of a primer VR2332-F1 is 0.9 mu M, the concentration of a primer VR2332-R1 is 0.9 mu M, and the concentration of a probe VR2332-P1 is 0.2 mu M.
In the method I or the method II or the method III, the reaction system of the digital RT-PCR can consist of 2 Xone-stepRT-ddPCR Supermix for probes, a primer LV-F1, a primer LV-R1, a probe LV-P1, a primer VR2332-F1, a primer VR2332-R1, a probe VR2332-P1 and a template.
In the method I or the method II or the method III, the reaction system of the digital RT-PCR can be composed of 2 Xone-stepRT-ddPCR Supermix for probes, a primer LV-F1, a primer LV-R1, a probe LV-P1, a primer VR2332-F1 and a primerVR2332-R1, Probe VR2332-P1, template and RNase Free dH2And (C) O.
In the method I or the method II or the method III, the reaction procedure of the digital RT-PCR can be as follows: reverse transcription is carried out for 10min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30sec and annealing at 55 ℃ for 60sec for 40 cycles; 10min at 98 ℃.
In the method I, the method II or the method III, the temperature rise and fall speed is set to be 2.5 ℃/sec in the reaction procedure of the digital RT-PCR.
The invention also protects a premixed solution which contains a primer LV-F1, a primer LV-R1, a probe LV-P1, a primer VR2332-F1, a primer VR2332-R1 and a probe VR 2332-P1; in the premix, the concentration of a primer LV-F1 is 1 mu M, the concentration of a primer LV-R1 is 1 mu M, the concentration of a probe LV-P1 is 2/9 mu M, the concentration of a primer VR2332-F1 is 1 mu M, the concentration of a primer VR2332-R1 is 1 mu M, and the concentration of a probe VR2332-P1 is 2/9 mu M. The premix functions as (c1), (c2) or (c3) as follows: (c1) identifying a porcine reproductive and respiratory syndrome virus european type and/or a porcine reproductive and respiratory syndrome virus american type; (c2) detecting whether the sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus; (c3) and detecting the content of the European type and/or the American type of the porcine reproductive and respiratory syndrome virus in the sample to be detected.
The invention also provides a kit comprising the pre-mix and microdroplet generating oil. The kit further comprises a negative control and a positive control; the negative control is RNase Free dH2O; the positive control is a solution containing total RNA of European type of porcine reproductive and respiratory syndrome virus and total RNA of American type of porcine reproductive and respiratory syndrome virus. The function of the kit is as follows (c1), (c2) or (c 3): (c1) identifying a porcine reproductive and respiratory syndrome virus european type and/or a porcine reproductive and respiratory syndrome virus american type; (c2) detecting whether the sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus; (c3) detecting the content of European type and/or American type of porcine reproductive and respiratory syndrome virus in a sample to be detectedAmount of the compound (A).
The invention also protects the application of the premix or the kit, which is (c1), (c2) or (c 3): (c1) identifying a porcine reproductive and respiratory syndrome virus european type and/or a porcine reproductive and respiratory syndrome virus american type; (c2) detecting whether the sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus; (c3) and detecting the content of the European type and/or the American type of the porcine reproductive and respiratory syndrome virus in the sample to be detected.
The kit further comprises a carrier carrying method IV and/or method V and/or method VI.
The method IV is a method for detecting whether the virus to be detected is the European type of porcine reproductive and respiratory syndrome virus and/or the American type of porcine reproductive and respiratory syndrome virus by digital RT-PCR, and comprises the following steps: taking total RNA of a virus to be detected or diluent thereof as template solution, mixing 2 mu L of template solution and 18 mu L of the premixed solution, placing the mixed solution and 70 mu L of microdroplet generation oil in a microdroplet generator to form microdroplets, then carrying out RT-PCR amplification, and then carrying out detection in a microdroplet analyzer; if the detection result based on the fluorescent group corresponding to the probe LV-P1 is positive, the virus to be detected is or is selected as the European porcine reproductive and respiratory syndrome virus, if the detection result based on the fluorescent group corresponding to the probe LV-P1 is negative, the virus to be detected is or is selected as the European non-porcine reproductive and respiratory syndrome virus; if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is positive, the virus to be detected is or is candidate to be the porcine reproductive and respiratory syndrome virus American type, and if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is negative, the virus to be detected is or is candidate to be the non-porcine reproductive and respiratory syndrome virus American type.
The method V is a method for detecting whether a sample to be detected contains the European type of the porcine reproductive and respiratory syndrome virus and/or the American type of the porcine reproductive and respiratory syndrome virus by digital RT-PCR, and comprises the following steps: taking total RNA of a sample to be detected or diluent thereof as template solution, mixing 2 mu L of template solution and 18 mu L of the premixed solution, placing the mixed solution and 70 mu L of microdroplet generation oil in a microdroplet generator to form microdroplets, then carrying out RT-PCR amplification, and then carrying out detection in a microdroplet analyzer; if the detection result based on the fluorescent group corresponding to the probe LV-P1 is positive and the sample to be detected contains the European porcine reproductive and respiratory syndrome virus, if the detection result based on the fluorescent group corresponding to the probe LV-P1 is negative and the sample to be detected does not contain the European porcine reproductive and respiratory syndrome virus; if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is positive, the sample to be detected contains the porcine reproductive and respiratory syndrome virus American type, and if the detection result based on the fluorescent group corresponding to the probe VR2332-P1 is negative, the sample to be detected does not contain the porcine reproductive and respiratory syndrome virus American type.
The method VI is a method for detecting the content of the European type and/or the American type of the porcine reproductive and respiratory syndrome virus in the sample to be detected by digital RT-PCR, and comprises the following steps: taking total RNA of a sample to be detected or diluent thereof as template solution, mixing 2 mu L of template solution and 18 mu L of the premixed solution, placing the mixed solution and 70 mu L of microdroplet generation oil in a microdroplet generator to form microdroplets, then carrying out RT-PCR amplification, and then carrying out detection in a microdroplet analyzer; obtaining the European content of the porcine reproductive and respiratory syndrome virus in a sample to be detected according to the number of the European positive droplets of the porcine reproductive and respiratory syndrome virus, wherein the European positive droplets of the porcine reproductive and respiratory syndrome virus are droplets with positive detection results based on fluorescent groups corresponding to a probe LV-P1; and obtaining the American type content of the porcine reproductive and respiratory syndrome virus in the sample to be detected according to the number of the American type positive droplets of the porcine reproductive and respiratory syndrome virus, wherein the American type positive droplets of the porcine reproductive and respiratory syndrome virus are droplets with positive detection results based on fluorescent groups corresponding to the probes VR 2332-P1.
In the method IV or the method V or the method VI, the reaction procedure of the digital RT-PCR can be as follows: reverse transcription is carried out for 10min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30sec and annealing at 55 ℃ for 60sec for 40 cycles; 10min at 98 ℃.
In the method IV, the method V or the method VI, the temperature rise and fall speed is set to be 2.5 ℃/sec in the reaction procedure of the digital RT-PCR.
Any one of the above digital RT-PCR may specifically be a droplet digital RT-PCR.
Any one of the above kits can be specifically a microdroplet digital RT-PCR absolute quantitative detection kit.
Any of the above digital RT-PCR specifically employs the QX200Droplet Digital PCR system.
Any of the above test samples can be a test porcine tissue sample, such as a porcine lung sample.
Any one of the viruses to be detected can be porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine circovirus type 2, pseudorabies virus or classical swine fever virus. Any of the porcine reproductive and respiratory syndrome virus described above may be porcine reproductive and respiratory syndrome virus American type or porcine reproductive and respiratory syndrome virus European type. Any of the porcine reproductive and respiratory syndrome virus American types described above may specifically be the VR-2332 strain. Any of the porcine reproductive and respiratory syndrome virus European types described above may specifically be the LV strain. Any one of the porcine parvoviruses can be particularly an NADL-2 strain. Any one of the porcine circovirus type 2 can be a 08TJ strain. Any of the pseudorabies viruses described above may in particular be the Bartha strain. Any one of the classical swine fever viruses described above may specifically be a C-strain.
The inventor designs and screens primer probe combinations capable of identifying European types and/or American types of the porcine reproductive and respiratory syndrome virus, further searches the concentrations of the primers and the probes, and finds the optimal working concentrations of the primers and the probes so as to improve the amplification efficiency of ddPCR.
The invention provides the micro-drop digital RT-PCR kit which has high sensitivity, high specificity, high accuracy and high precision and can realize accurate quantification, is used for detecting the European type and/or the American type of the porcine reproductive and respiratory syndrome virus, can directly quantify, does not need a standard curve, has simple and convenient operation and accurate and reliable result, and is particularly suitable for field detection. The invention has great application value for the prevention and control of European type and/or American type of the porcine reproductive and respiratory syndrome virus, and is beneficial to controlling epidemic situation from the source and effectively preventing large-scale outbreak of the porcine epidemic disease.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The digital PCR in the examples was performed using a QX200Dropletdigital PCR system (comprising two instruments: a QX200droplet generator and a QX200droplet analyzer) from Bio-Rad Laboratories, Inc. The heat sealer used in the examples was PX1TMPCR Heat sealer, the microdroplet generation card used in the examples is DG8TMThe microtropGeneration card (8X 3), the base used in the examples was the generation card base, and the microtropGeneration oil used in the examples was the microtropGeneration oil (for probes), all of which are products of Bio-Rad Laboratories, Inc. 2 Xone-step RT-ddPCR Supermix for probes, a product of Bio-Rad Laboratories, Inc.
The American strain of porcine reproductive and respiratory syndrome virus used in the examples isVR-2332TMSequence number in GENBANK is U87392. The European strain of porcine reproductive and respiratory syndrome virus used in the examples was LV (Lelystad virus) and the sequence number in GENBANK was M96262. The porcine parvovirus used in the examples was a porcine parvovirus strain NADL-2,
VR-742TMand the sequence number in GENBANK is KF 913351. The porcine circovirus type 2 strain used in the examples was 08TJ, and the sequence number in GENBANK was HQ 395021. The pseudorabies virus strain used in the examples was Bartha and the sequence number in GENBANK was JF 797217. Fruit of Chinese wolfberryThe classical swine fever virus strain used in the examples was C-strain, sequence number Z46258 in GENBANK.
Example 1 design and screening of primers and probes
Design and screening of primers and probes for detecting European strain of porcine reproductive and respiratory syndrome virus
Four primers and two probes are designed and preliminarily screened through a large number of sequence acquisition, analysis, comparison and preliminary experiments, and the nucleotide sequences are as follows:
LV-F1 (SEQ ID NO: 1 of the sequence Listing): 5 '-GCTGCMGGTTGCYCATACA-3';
LV-R1 (SEQ ID NO: 2 of the sequence Listing): 5 '-CAATCGHGGCCATTCAC-3';
LV-P1 (SEQ ID NO: 3 of the sequence Listing): 5 '-tgcgbctgatcgcgtdacttc-3'; .
LV-F2：5’-TGCTGCMGGTTGCTCATAC-3’；
LV-R2：5’-TCAATCGCGKCCATTCA-3’；
LV-P2：5’-CGCMTGATTCGCGTGACTTCTAVATCC-3’。
In the above nucleotide sequences, M represents A or C, Y represents C or T, H represents A, T or C, B represents G, T or C, D represents G, A or T, M represents A or C, K represents G or T, and V represents G, A or C.
LV-F1 and LV-F2 are both upstream primers, LV-R1 and LV-R2 are both downstream primers, and LV-P1 and LV-P2 are both probes. LV-P1 was labeled at the 5 'end with a fluorophore FAM and at the 3' end with a fluorescence quencher BHQ 1. LV-P2 was labeled at the 5 'end with a fluorophore FAM and at the 3' end with a fluorescence quencher BHQ 1.
Screening of primer probe set for detecting European strain of porcine reproductive and respiratory syndrome virus
Randomly combining the four primers obtained by screening in the step one and the two probes to form eight primer probe sets: LV-F1R1P1 consisting of LV-F1, LV-R1 and LV-P1, LV-F1R1P2 consisting of LV-F1, LV-R1 and LV-P2, LV-F1R2P1 consisting of LV-F1, LV-R2 and LV-P1, LV-F1R2P2 consisting of LV-F1, LV-R2 and LV-P2, LV-F1R2P2, LV-R1 and LV-P1, LV-F2R1P1 consisting of LV-F2, LV-R1 and LV-P2, LV-F2R1P2 consisting of LV-F2, LV-R2 and LV-P1, LV-F2R2P1 consisting of LV-F2, LV-R2 and LV-P2, and LV-F2R2P 2.
1. And (3) extracting total RNA from European strains of the porcine reproductive and respiratory syndrome virus.
2. And (3) taking the total RNA obtained in the step (1) as a template, and respectively adopting each primer probe group to perform real-time fluorescent quantitative RT-PCR.
Reaction system for RT-qPCR (20. mu.L):
probe qPCR dUTP Master Mix 10. mu.L, GoScript RT Mix for 1-step RT-QPCR 0.5. mu.L, 10. mu.M forward primer solution 1.2. mu.L, 10. mu.M reverse primer solution 1.2. mu.L, 10. mu.M Probe solution 0.6. mu.L, template 2. mu.L, and balance water.
Reaction procedure for RT-qPCR: 10min at 45 ℃; 2min at 95 ℃; at 95 ℃ for 15s and 60 ℃ for 1min, and 40 cycles.
Real-time quantitative fluorescence RT-PCR was performed using the primer probe sets LV-F1R1P1, with a peak value of 1000000 for the &lTt transformation &l Tt/T &gTt Rn and 300000 for the real-time quantitative fluorescence RT-PCR using seven additional primer probe sets, with a peak value of 300000 for the &lTtT transformation &/T &gTt Rn, and the signal value of the amplification curve was strongest using the primer probe sets LV-F1R1P 1.
Design and screening of primers and probes for detecting porcine reproductive and respiratory syndrome virus American strain
Four primers and two probes are designed and preliminarily screened through a large number of sequence acquisition, analysis, comparison and preliminary experiments, and the nucleotide sequences are as follows:
VR2332-F1 (SEQ ID NO: 5 of the sequence Listing): 5 '-TTAGTYTGCCTACGCWTCATACHGT-3';
VR2332-R1 (SEQ ID NO: 6 of the sequence Listing): 5 '-TGCYAGCMCATCATSCT-3';
VR2332-P1 (SEQ ID NO: 7 of the sequence Listing): 5 '-CGYCTGATCHGCGTCRCAGC-3';
VR2332-F2：5’-TYTAGTTTGCCTACGCATCATACTG-3’；
VR2332-R2：5’-ATGCCAGCHCATCATGCT-3’；
VR2332-P2：5’-ATCCGCGTCACAGCATCACBCTC-3’。
in the above nucleotide sequences, Y represents C or T, W represents A or T, H represents A, T or C, M represents A or C, S represents G or C, R represents A or G, B represents G, T or C.
VR2332-F1 and VR2332-F2 are both upstream primers, VR2332-R1 and VR2332-R2 are both downstream primers, and VR2332-P1 and VR2332-P2 are both probes. The 5 'end of VR2332-P1 is labeled with a fluorescent group HEX, and the 3' end is labeled with a fluorescence quenching group BHQ 1. The 5 'end of VR2332-P2 is labeled with a fluorescent group HEX, and the 3' end is labeled with a fluorescence quenching group BHQ 1.
Screening of primer probe set for detecting porcine reproductive and respiratory syndrome virus American strain
Randomly combining the four primers obtained by screening in the step one and the two probes to form eight primer probe sets: a primer probe group VR2332-F1R1P1 consisting of VR2332-F1, VR2332-R1 and VR2332-P1, a primer probe group VR2332-F1R1P2 consisting of VR2332-F1, VR2332-R1 and VR2332-P2, a primer probe group VR2332-F1R2P1 consisting of VR2332-F1, VR2332-R2 and VR2332-P1, a primer probe group 2332-F1R2P 8672 consisting of VR2332-F1, VR2332-R2 and VR2332-P2, a primer probe group VR2332-F2R1P2 consisting of VR2332-F2, VR2332-R2 and VR2332-P2, a primer probe group VR 2332-F2-F1P 23372 consisting of VR 2332-F2332-R2 and VR2332-P2, a primer probe group VR 2332-F2332P 23372 consisting of VR 2332-F2-F2332-P23372, a primer probe group VR 2332-F2-F2332-P2 and VR2332-P2, a primer probe set VR2332-F2R2P2 consisting of VR2332-F2, VR2332-R2 and VR 2332-P2.
1. And (3) taking the American strain of the porcine reproductive and respiratory syndrome virus, and extracting total RNA.
2. And (3) taking the total RNA obtained in the step (1) as a template, and respectively adopting each primer probe group to perform real-time fluorescent quantitative RT-PCR.
Reaction system for RT-qPCR (20. mu.L):
probe qPCR dUTP Master Mix 10. mu.L, GoScript RT Mix for 1-step RT-QPCR 0.5. mu.L, 10. mu.M forward primer solution 1.2. mu.L, 10. mu.M reverse primer solutionSolution 1.2. mu.L, 10. mu.M probe solution 0.6. mu.L, template 2. mu.L, and balance water.
Reaction procedure for RT-qPCR: 10min at 45 ℃; 2min at 95 ℃; at 95 ℃ for 15s and 60 ℃ for 1min, and 40 cycles.
Real-time quantitative fluorescence RT-PCR was performed using the primer probe set VR2332-F1R1P1, the peak value of &lTtT transfer = delta "&Δ &lTt/T &gTt Rn was 1000000, real-time quantitative fluorescence RT-PCR was performed using seven additional primer probe sets, the peak value of &lTtTtransfer &Δ &lTt/T &gTt was 300000-500000, and the signal value of the amplification curve was strongest using the primer probe set VR2332-F1R1P 1.
Example 2 optimization of relevant reaction parameters in digital PCR
First, optimization of annealing temperature
1. And (3) extracting total RNA from European strains of the porcine reproductive and respiratory syndrome virus.
2. And (3) taking the American strain of the porcine reproductive and respiratory syndrome virus, and extracting total RNA.
3. And (3) mixing the total RNA obtained in the step (1) and the total RNA obtained in the step (2) in equal mass to obtain mixed RNA.
4. And (3) performing digital RT-PCR by using the mixed RNA obtained in the step (3) as a template and adopting a primer combination consisting of a primer probe group LV-F1R1P1 and a primer probe group VR2332-F1R1P 1.
(1) The following system (20. mu.L) was prepared: 10 μ L of 2 Xone-step RT-ddPCR Supermix for probes, 1 μ L LV-F1, 1 μ L LV-R1, 0.5 μ L LV-P1, 1 μ L VR2332-F1, 1 μ L VR2332-R1, 0.5 μ L VR2332-P1, 2 μ L template, 3 μ L RNase FreedH2And O. In the system, the concentration of the forward primer was 0.5. mu.M, the concentration of the reverse primer was 0.5. mu.M, and the concentration of the probe was 0.25. mu.M.
(2) Fixing the microdroplet generation card on a base, adding 20 mu L of the system prepared in the step (1) into each hole in 8 holes in the middle row, adding 70 mu L of microdroplet generation oil into each hole in 8 holes in the bottom row, and then placing the base fixed with the microdroplet generation card in a microdroplet generator to form microdroplets (the microdroplets are generated in the 8 holes in the top row of the microdroplet generation card).
(3) After completion of step (2) using a 96-well plate (designated as 96-well plate I), 40. mu.L of each well from the top row of 8 wells of the droplet generation card was added in a one-to-one correspondence to the 8 wells of the 96-well plate I, and the membrane was sealed with a heat sealer. After completion of step (2) using a 96-well plate (designated as 96-well plate II), 40. mu.L of each well from the top row of 8 wells of the microdroplet card was added in a one-to-one correspondence to 8 wells of the 96-well plate II, and the membrane was sealed with a heat sealer. After completion of step (2) using a 96-well plate (designated as 96-well plate III), 40. mu.L of each well from the top row of 8 wells of the droplet generation card was added in a one-to-one correspondence to 8 wells of the 96-well plate III, and the membrane was sealed with a heat sealer. After completion of step (2) using a 96-well plate (designated as 96-well plate IV), 40. mu.L of each well from the top row of 8 wells of the droplet generation card was added in a one-to-one correspondence to 8 wells of the 96-well plate IV, and the membrane was sealed with a heat sealer. After completion of step (2) using a 96-well plate (designated 96-well plate V), 40. mu.L of each well from the top row of 8 wells of the droplet generation card was added in a one-to-one correspondence to 8 wells of the 96-well plate V, and the membrane was sealed with a heat sealer. And (3) taking a 96-well plate (named as a 96-well plate VI), after the step (2) is completed, adding 40 mu L of each of 8 wells in a row at the top of the droplet generation card into the 8 wells of the 96-well plate VI in a one-to-one correspondence manner, and sealing the membrane by using a heat sealing instrument.
(4) And (4) placing each 96-well plate which completes the step (3) in a different PCR instrument for RT-PCR amplification.
RT-PCR amplification procedure: reverse transcription is carried out for 10min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30sec and annealing for 60sec for 40 cycles; 10min at 98 ℃. The ramp rate used was set at 2.5 deg.C/sec.
And (3) setting the following annealing temperatures in sequence for the PCR instrument from the 96-well plate I to the 96-well plate VI: 52.0 deg.C, 54.0 deg.C, 55.0 deg.C, 56.0 deg.C, 58.0 deg.C, 60.0 deg.C.
(5) And (4) after the step (4) is finished, taking a 96-well plate, and detecting in a microdroplet analyzer, wherein the microdroplet which is positive to the European type of the porcine reproductive and respiratory syndrome virus shows blue fluorescence, and the microdroplet which is positive to the American type of the porcine reproductive and respiratory syndrome virus shows green fluorescence.
The annealing temperature of 52.0 ℃ is adopted, and in each microliter of the template, the European type of the porcine reproductive and respiratory syndrome virus is detected to be 1013copies, and the American type of the porcine reproductive and respiratory syndrome virus is detected to be 1020 copies. The European type of porcine reproductive and respiratory syndrome virus was detected as 1306copies and the American type of porcine reproductive and respiratory syndrome virus was detected as 1298copies per microliter of template using an annealing temperature of 54.0 ℃. The annealing temperature of 55.0 ℃ is adopted, and in each microliter of template, the European type of the porcine reproductive and respiratory syndrome virus is detected to be 1325copies, and the American type of the porcine reproductive and respiratory syndrome virus is detected to be 1310 copies. The annealing temperature of 56.0 ℃ is adopted, and in each microliter of the template, the European type of the porcine reproductive and respiratory syndrome virus is detected to be 1316copies, and the American type of the porcine reproductive and respiratory syndrome virus is detected to be 1309 copies. The annealing temperature of 58.0 ℃ is adopted, and in each microliter of the template, the European type of the porcine reproductive and respiratory syndrome virus is detected as 1116copies, and the American type of the porcine reproductive and respiratory syndrome virus is detected as 1108 copies. The annealing temperature of 60.0 ℃ is adopted, and in each microliter of template, the detection value of the European type of the porcine reproductive and respiratory syndrome virus is 1145copies, and the detection value of the American type of the porcine reproductive and respiratory syndrome virus is 1132 copies.
The annealing temperature of 55.0 ℃ is adopted, and the detected value is closest to the actual value.
The result shows that the annealing temperature can be selected to be 54-56 ℃, and the optimal annealing temperature is 55 ℃.
Second, optimization of primer and probe concentration
1. And (3) extracting total RNA from European strains of the porcine reproductive and respiratory syndrome virus.
2. And (3) taking the American strain of the porcine reproductive and respiratory syndrome virus, and extracting total RNA.
3. And (3) mixing the total RNA obtained in the step (1) and the total RNA obtained in the step (2) in equal mass to obtain mixed RNA.
4. And (3) performing digital RT-PCR by using the mixed RNA obtained in the step (3) as a template and adopting a primer combination consisting of a primer probe group LV-F1R1P1 and a primer probe group VR2332-F1R1P 1.
(1) Different systems were prepared, see in particular table 1 (the numbers in the table are the added volumes of the individual components, in μ L). The concentration of the forward primer, the concentration of the reverse primer and the concentration of the probe used in the preparation system were all 10. mu.M.
TABLE 1
| System 1 | System 2 | System 3 | System 4 | |
| LV-F1 | 0.8 | 1.2 | 1.6 | 1.8 |
| LV-R1 | 0.8 | 1.2 | 1.6 | 1.8 |
| LV-P1 | 0.15 | 0.25 | 0.35 | 0.4 |
| VR2332-F1 | 0.8 | 1.2 | 1.6 | 1.8 |
| VR2332-R1 | 0.8 | 1.2 | 1.6 | 1.8 |
| VR2332-P1 | 0.15 | 0.25 | 0.35 | 0.4 |
| 2×One-step RT-ddPCR Supermix for probes | 10 | 10 | 10 | 10 |
| RNAse Free dH2O | 4.5 | 2.7 | 0.9 | 0 |
| Form panel | 2 | 2 | 2 | 2 |
| Total volume | 20 | 20 | 20 | 20 |
(2) Fixing the microdroplet generation card on a base, adding 20 mu L of the system prepared in the step (1) into each hole in 8 holes in the middle row, adding 70 mu L of microdroplet generation oil into each hole in 8 holes in the bottom row, and then placing the base fixed with the microdroplet generation card in a microdroplet generator to form microdroplets (the microdroplets are generated in the 8 holes in the top row of the microdroplet generation card).
(3) And (3) taking a 96-well plate, finishing the step (2), adding 40 mu L of each well from 8 wells in the top row of the microdroplet generation card into 8 wells of the 96-well plate in a one-to-one correspondence manner, and sealing the membrane by using a heat sealing instrument.
(4) And (4) placing each 96-well plate which completes the step (3) in a different PCR instrument for RT-PCR amplification.
RT-PCR amplification procedure: reverse transcription is carried out for 10min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30sec and annealing at 55 ℃ for 60sec for 40 cycles; 10min at 98 ℃. The ramp rate used was set at 2.5 deg.C/sec.
(5) And (4) after the step (4) is finished, taking a 96-well plate, and detecting in a microdroplet analyzer, wherein the microdroplet which is positive to the European type of the porcine reproductive and respiratory syndrome virus shows blue fluorescence, and the microdroplet which is positive to the American type of the porcine reproductive and respiratory syndrome virus shows green fluorescence.
By adopting the system 1, in each microliter of the template, the European type of the porcine reproductive and respiratory syndrome virus is detected to be 3860copies, and the American type of the porcine reproductive and respiratory syndrome virus is detected to be 3840 copies. With the system 2, in each microliter of the template, the European type of porcine reproductive and respiratory syndrome virus was detected as 3990copies, and the American type of porcine reproductive and respiratory syndrome virus was detected as 3960 copies. By adopting the system 3, in each microliter of template, the detection value of the European type of the porcine reproductive and respiratory syndrome virus is 4090copies, and the detection value of the American type of the porcine reproductive and respiratory syndrome virus is 4070 copies. By adopting the system 4, in each microliter of template, the European type detection value of the porcine reproductive and respiratory syndrome virus is 4280copies, and the American type detection value of the porcine reproductive and respiratory syndrome virus is 4265 copies.
With system 4, the measured value is closest to the actual value.
The results show that the optimal primer probe concentrations in the reaction system are as follows: L-F10.9 μ M, LV-R10.9 μ M, LV-P10.2 μ M, VR 2332-F10.9 μ M, VR 2332-R10.9 μ M, VR 2332-P10.2 μ M.
Example 3 preparation of the kit
Preparation of respective reagents
The solution A is a one-step ddPCR probe method premix solution. The composition of solution A per 900. mu.L was as follows: mu.L of 2 Xone-step RT-ddPCR Supermix for probes, 90. mu.L of LV-F1 solution (the concentration of LV-F1 in LV-F1 solution is 10. mu.M), 90. mu.L of VR2332-F1 solution (the concentration of VR2332-F1 in VR2332-F1 solution is 10. mu.M), 90. mu.L of LV-R1 solution (the concentration of LV-R1 in LV-R1 solution is 10. mu.M), 90. mu.L of VR2332-R1 solution (the concentration of VR2332-R1 in VR2332-R1 solution is 10. mu.M), 20. mu.L of LV-P1 solution (the concentration of LV-P1 in LV-P1 solution is 10. mu.M), and 20. mu.L of VR2332-P1 solution (the concentration of VR2332-P1 in VR2332-P1 solution is 10. mu.M).
Solution B is a microdroplet generating oil.
Solution C is a positive control. Preparation method of solution C: extracting total RNA of European type of porcine reproductive and respiratory syndrome virus, extracting total RNA of American type of porcine reproductive and respiratory syndrome virus, mixing the two total RNAs with equal mass, and diluting with Tris-EDTA buffer (pH8.0, 0.01M) to make the concentration of the two RNAs 10000 copies/microliter.
Solution D is a negative control. Solution D is RNase Free dH2O。
Second, assembly of microdroplet digital PCR absolute quantitative detection kit
The kit comprises the following components: and respectively and independently packaging the solution A, the solution B, the solution C and the solution D.
Third, using method of kit
The sample to be detected is virus to be detected or pig tissue to be detected, and the pig tissue can be pig lung.
1. Extracting total RNA of a sample to be detected, and taking the total RNA or a diluent thereof as a template solution.
2. mu.L of solution A was taken and 2. mu.L of template solution was added. An equal volume of solution D was used as a negative control treatment instead of the template solution. An equal volume of solution C was used as a positive control treatment instead of the template solution.
3. The card is fixed on a base, 20. mu.L of the system prepared in step 2 is added into each hole in the 8 holes in the middle row, 70. mu.L of solution B is added into each hole in the 8 holes in the bottom row, and then the base with the card is placed in a droplet generator to form droplets (the droplets are generated in the 8 holes in the top row of the card).
4. After completing step 3, 40. mu.L of each of the 8 wells from the top row of the microtiter plate was added in a one-to-one correspondence to the 8 wells of the 96-well plate, and the membrane was sealed with a heat sealer.
5. And (4) placing the 96-well plate which completes the step 4 in a PCR instrument for RT-PCR amplification.
RT-PCR amplification procedure: reverse transcription is carried out for 10min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30sec and annealing at 55 ℃ for 60sec for 40 cycles; 10min at 98 ℃. The ramp rate used was set at 2.5 deg.C/sec.
6. After step 5, taking a 96-well plate, detecting in a microdroplet analyzer, wherein the microdroplet which is positive to the European type of the porcine reproductive and respiratory syndrome virus shows blue fluorescence, and the microdroplet which is positive to the American type of the porcine reproductive and respiratory syndrome virus shows green fluorescence.
The result is credible when the following four conditions are met, wherein in ① microliter positive control, the detection value of the European type of the porcine reproductive and respiratory syndrome virus is 10000 +/-100 copy numbers, in ② microliter positive control, the detection value of the American type of the porcine reproductive and respiratory syndrome virus is 10000 +/-100 copy numbers, in ③ microliter negative control, the detection value of the European type of the porcine reproductive and respiratory syndrome virus is 0, in ④ microliter negative control, the detection value of the American type of the porcine reproductive and respiratory syndrome virus is 0, if a droplet of the European type positive of the porcine reproductive and respiratory syndrome virus is detected in the template, the European type of the porcine reproductive and respiratory syndrome virus is contained in the sample to be detected, the copy number of the European type of the porcine reproductive and respiratory syndrome virus can be obtained according to the number of positive droplets, if no droplet of the European type positive of the porcine reproductive and respiratory syndrome virus is detected in the template, the European type positive droplet of the porcine reproductive and respiratory syndrome virus in the sample to be detected is not contained in the template, and the European type of the porcine reproductive and respiratory syndrome can be obtained according to the number of the porcine reproductive and respiratory syndrome in the droplet of the American type of the porcine reproductive and respiratory syndrome detected in the template.
Example 4 specificity test
The samples to be tested are respectively as follows: 3D4/21 porcine lung cell strain, porcine parvovirus, porcine circovirus type 2, pseudorabies virus and classical swine fever virus.
The kit prepared in example 3 was used, and detection was performed according to the method of use of the kit.
Each sample to be tested is negative.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Chinese animal epidemic prevention control center
<120> European and American double microdroplet digital PCR absolute quantitative detection test for porcine reproductive and respiratory syndrome virus
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Claims (6)
1. A primer probe combination, which consists of a primer probe group LV-F1R1P1 and a primer probe group VR2332-F1R1P 1;
the primer probe group LV-F1R1P1 consists of a primer LV-F1, a primer LV-R1 and a probe LV-P1; the primer LV-F1 is a single-stranded DNA molecule shown in a sequence 1 in a sequence table; the primer LV-R1 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table; one end of the probe LV-P1 is provided with a fluorescence reporter group, the other end is provided with a fluorescence quenching group, and the nucleotide sequence is shown as a sequence 3 in a sequence table;
the primer probe group VR2332-F1R1P1 consists of a primer VR2332-F1, a primer VR2332-R1 and a probe VR 2332-P1; the primer VR2332-F1 is a single-stranded DNA molecule shown as a sequence 4 in a sequence table; the primer VR2332-R1 is a single-stranded DNA molecule shown as a sequence 5 in a sequence table; one end of the probe VR2332-P1 is provided with a fluorescence reporter group, the other end is provided with a fluorescence quenching group, and the nucleotide sequence is shown as sequence 6 in the sequence table;
the probe LV-P1 and the probe VR2332-P1 have fluorescent reporter groups of different colors.
2. The primer probe combination of claim 1, which is used as (b1), (b2) or (b 3):
(b1) preparing a kit for identifying porcine reproductive and respiratory syndrome virus European type and/or porcine reproductive and respiratory syndrome virus American type;
(b2) preparing a kit for detecting whether a sample to be detected contains the European type of porcine reproductive and respiratory syndrome virus and/or the American type of porcine reproductive and respiratory syndrome virus;
(b3) preparing a kit for detecting the European type and/or American type content of the porcine reproductive and respiratory syndrome virus in a sample to be detected.
3. A kit comprising the primer probe combination of claim 1.
4. A premix for detecting European type porcine reproductive and respiratory syndrome virus and/or American type porcine reproductive and respiratory syndrome virus by digital RT-PCR comprising the primer LV-F1, the primer LV-R1, the probe LV-P1, the primer VR2332-F1, the primer VR2332-R1 and the probe VR2332-P1 as set forth in claim 1; in the premix, the concentration of a primer LV-F1 is 1 mu M, the concentration of a primer LV-R1 is 1 mu M, the concentration of a probe LV-P1 is 2/9 mu M, the concentration of a primer VR2332-F1 is 1 mu M, the concentration of a primer VR2332-R1 is 1 mu M, and the concentration of a probe VR2332-P1 is 2/9 mu M.
5. A kit for detecting porcine reproductive and respiratory syndrome virus european type and/or porcine reproductive and respiratory syndrome virus american type by digital RT-PCR comprising the premix and the microdroplet generating oil of claim 4.
6. The kit of claim 5, wherein: the kit further comprises a negative control and a positive control; the negative control is RNase Free dH2O; the positive control is a solution containing total RNA of European type of porcine reproductive and respiratory syndrome virus and total RNA of American type of porcine reproductive and respiratory syndrome virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710845507.0A CN107475451B (en) | 2017-09-19 | 2017-09-19 | European and American dual microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome virus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710845507.0A CN107475451B (en) | 2017-09-19 | 2017-09-19 | European and American dual microdroplet digital PCR absolute quantitative detection kit for porcine reproductive and respiratory syndrome virus |
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| CN108977577A (en) * | 2018-06-28 | 2018-12-11 | 暨南大学 | Primer and probe and its kit and method based on digital pcr technology detection PRRSV Europe class virus |
| CN112779352B (en) * | 2019-11-11 | 2023-07-21 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Dual digital PCR detection technology for swine fever and African swine fever and special kit thereof |
| CN112195273A (en) * | 2020-09-08 | 2021-01-08 | 华南农业大学 | Kit for detecting porcine reproductive and respiratory syndrome virus by combining centrifugal microfluidic chip with loop-mediated isothermal amplification technology |
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