CN114540546A

CN114540546A - Primer probe set, kit and detection method for PRRSV and CSFV double fluorescence quantitative PCR detection

Info

Publication number: CN114540546A
Application number: CN202210140285.3A
Authority: CN
Inventors: 赵阳; 魏启超; 武守霖; 孙彩云; 袁丹阳; 董雪莹
Original assignee: Harbin Zhongke Gene Technology Co ltd
Current assignee: Harbin Zhongke Gene Technology Co ltd
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-05-27

Abstract

The invention relates to a primer probe set, a kit and a detection method for double fluorescence quantitative PCR detection of PRRSV and CSFV, belonging to the technical field of molecular biology. In order to further improve the accuracy of the PRRSV and CSFV double fluorescence quantitative PCR joint inspection, the invention provides a primer probe set for PRRSV and CSFV double fluorescence quantitative PCR detection, the PRRSV-specific primer probe set and the CSFV-specific primer probe set are designed by taking ORF6 gene of PRRSV and 5' UTR non-coding region of CSFV as amplification target regions, the conservatism and stability of the detection gene locus are ensured, the sequence structure of the primer probe set has no interaction, the amplification efficiency and the binding rate are high, and the detection sensitivity is very high. The detection kit provided by the invention has no cross reaction with similar pathogens, has higher specificity and accuracy, and can meet the requirement of large-scale rapid identification and detection.

Description

Primer probe set, kit and detection method for PRRSV and CSFV double fluorescence quantitative PCR detection

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a primer probe set, a kit and a detection method for PRRSV and CSFV double fluorescence quantitative PCR detection.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), also known as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), is mainly caused by PRRSV, can cause severe Reproductive failure of pregnant sows and Respiratory disturbance of pigs of various ages, and is a Porcine infectious disease which seriously affects the economic benefit of breeding. Classical Swine Fever (CSF) is mainly caused by CSFV, which causes high mortality and morbidity of Swine herds and destructive economic loss, and is classified as a class A infectious disease by OIE (animal health organization) in the world.

Since porcine reproductive and respiratory syndrome and classical swine fever can cause porcine reproductive disorders to various degrees and are often mixed with infections, accurate diagnosis is difficult to make only by clinical symptoms, pathological changes and epidemiological investigation, especially at early or latent stages of onset. Real-time fluorescent quantitative PCR, RT-PCR, is widely used in gene expression level analysis, qualitative and quantitative detection of pathogen genes and other fields due to its advantages of strong specificity, high sensitivity, high speed and the like, and has become the main method for rapid detection of viral nucleic acids at present.

When RT-PCR is applied to simultaneously joint detection of a plurality of viruses, if interaction exists between the primer sequence structures and the probe sequence structures of different viruses, negative influence is easily caused on the detection sensitivity and accuracy, and the detection sensitivity is reduced and the result is inaccurate. Meanwhile, factors such as rapid virus mutation, multiple genotypes and serotypes and the like also influence the sensitivity and accuracy of RT-PCR joint detection. The invention with the application number of CN201110146877.8 discloses a method for detecting the pestivirus of swine fever and the blue-ear disease virus by the double-color fluorescence quantitative PCR (polymerase chain reaction) combined detection and a kit thereof, which can realize the simultaneous detection of the pestivirus of swine and the reproductive and respiratory syndrome virus of swine in the same sample to be detected; however, the gene locus for detecting the porcine reproductive and respiratory syndrome is located in ORF5 gene, and the gene has high mutation and homologous recombination. The invention patent with application number CN201510191676.8 triple fluorescence RT-PCR detection reagent for African swine fever virus, swine fever virus and porcine reproductive and respiratory syndrome virus and the method and the application thereof has the advantages that the detection site of PRRSV is NSP2, the conservation of the site is low, even in highly pathogenic porcine reproductive and respiratory syndrome, a large amount of NSP2 genes are deleted. If the detected gene locus is mutated or deleted, a false negative detection result can be even directly caused, the specificity of the NSP2 variant strain is reduced, and misdirection is caused to the definite diagnosis of the sick pig.

Therefore, when performing RT-PCR synchronous joint inspection of various viruses, the selection of the detection gene locus and whether the primer probe set sequence structures of different viruses interact with each other are the key to determine the detection accuracy, but combining a suitable detection gene locus and a stable primer probe set sequence structure is not obvious from the gene sequence of the virus, and is still a technical difficulty to be solved in the field.

Disclosure of Invention

In order to further improve the accuracy of the PRRSV and CSFV double RT-PCR joint detection, the invention provides a primer probe set, a kit and a detection method for PRRSV and CSFV double fluorescent quantitative PCR detection.

The technical scheme of the invention is as follows:

a primer probe set for dual fluorescent quantitative PCR detection of PRRSV and CSFV, comprising a primer probe set for PRRSV and a primer probe set for CSFV;

the primer probe set aiming at the PRRSV comprises a primer and a probe with the following nucleotide sequences:

forward primer PRRSV-D1-F: 5'-ACCTGGAAATTCATCACCTC-3', respectively;

reverse primer PRRSV-D1-R: 5'-CGACAAATGCGTGGTTATCA-3', respectively;

probe PRRSV-Probe: 5 '-FAM-TGCTAGGCCGCAAGTACATTC-BHQ 1-3';

the primer probe group aiming at CSFV comprises a primer and a probe with the following nucleotide sequences:

forward primer CSFV-D155-18F: 5'-GGGTGGTCTAAGTCCTGA-3', respectively;

reverse primer CSFV-D325-18R: 5'-CTAATAGTGGGCCTCTGC-3', respectively;

probe CSFV-Probe: 5 '-ROX-CAGTAGTTCGACGTGAGCAGAAG-BHQ 2-3'.

A kit for double fluorescent quantitative PCR detection of PRRSV and CSFV comprises a forward primer PRRSV-D1-F, a reverse primer PRRSV-D1-R, a Probe PRRSV-Probe, a forward primer CSFV-D155-18F, a reverse primer CSFV-D325-18R and a Probe CSFV-Probe provided by the invention.

Furthermore, the kit also comprises DNA polymerase, reverse transcriptase mix, a negative control substance, a PRRSV and CSFV positive control substance and a PCR buffer solution containing dNTP.

Further, the DNA polymerase is preferably TaKaRa Ex Taq HS (5U/. mu.l) commercially available.

Further, the reverse transcriptase cocktail contains an RNase inhibitor, M-MuLV reverse transcriptase and a thermostable DNA polymerase, preferably the commercially available PrimeScript RT Enzyme Mix II.

Further, the PRRSV and CSFV positive control product is a mixture of PRRSV virus positive plasmid and CSFV virus positive plasmid, and the concentration of the PRRSV virus positive plasmid and the CSFV virus positive plasmid in the mixture is 2.4 multiplied by 10^-4ng/ul, the negative reference substance is sterile DEPC-H₂O。

Furthermore, the PRRSV virus positive plasmid is a recombinant plasmid constructed by connecting a product obtained by amplifying a target sequence by taking a nucleotide sequence shown as SEQ ID No.7 in a PRRSV ORF6 gene sequence conserved fragment and a vector, and a primer pair used for amplification has the following nucleotide sequence:

PRRSV-SF：5′-TTTCAGCGGAACAATGGGGTC-3′；

PRRSV-SR：5′-TTTCTGCCACCCAACACGAGG-3′；

the positive plasmid of the CSFV virus is a recombinant plasmid constructed by connecting a product obtained by amplifying a target sequence by taking a nucleotide sequence shown as SEQ ID No.10 in a CSFV 5' UTR gene sequence conserved fragment and a carrier, and the primer pair used for amplification has the following nucleotide sequence:

CSFV-S149-18F；5′-CTCCCTGGGTGGTCTAAG-3′；

CSFV-S729-18R；5′-GCTACCTGTCACCCTACC-3′。

further, the PCR Buffer solution containing dNTP comprises Buffer solution, magnesium chloride and dNTP, and is preferably 2X One Step RT-PCR Buffer III sold on the market.

A method for double fluorescent quantitative PCR detection of PRRSV and CSFV comprises the following steps:

step one, extracting a genome of a sample to be detected, using the obtained genome RNA as a template, performing double RT-PCR amplification by using the primer probe set of claim 1, and detecting a fluorescent signal when each cycle of extension is finished;

and step two, analyzing the PCR product obtained by amplification in the step one, and judging whether the sample to be detected contains PRRSV and/or CSFV or not based on the specific amplification curve generation condition and Ct value.

Further, the double RT-PCR amplification reaction system in the step one contains forward primer PRRSV-D1-F, reverse primer PRRSV-D1-R, forward primer CSFV-D155-18F and reverse primer CSFV-D325-18R with final concentration of 0.2 mu M, Probe PRRSV-Probe and Probe CSFV-Probe with final concentration of 0.4 mu M, PCR buffer solution 10 mu l, DNA polymerase 0.4 mu l of 5U/mu l, reverse transcription mixing enzyme 0.4 mu l, amplification template 2 mu l, RNase Free dH₂O make up to a total volume of 20. mu.l.

Further, the dual RT-PCR amplification reaction system of the first step is shown in Table 1:

TABLE 1

Further, the procedure of the double fluorescence quantitative PCR amplification reaction in the first step is a first stage, reverse transcription is carried out for 5min at 42 ℃, pre-denaturation is carried out for 10s at 95 ℃, and 1 cycle is carried out; the second stage, 95 ℃ for 5s and 60 ℃ for 34s, for 40 cycles.

Further, the probe detection mode of the step one fluorescence signal detection is set as follows: the fluorescent mode PRRSV is FAM/NONE double-labeling mode, and CSFV is ROX/NONE double-labeling mode.

Further, the determination method of the detection result in the second step is as follows:

if specific amplification curves appear in both the FAM/NONE double-labeling mode and the ROX/NONE double-labeling mode, and the Ct value is less than or equal to 36.0, determining that PRRSV and CSFV exist in the sample to be detected;

if a specific amplification curve appears only in the FAM/NONE double-labeling mode and the Ct value is less than or equal to 36.0, judging that the PRRSV exists in the sample to be detected;

if a specific amplification curve appears only in the ROX/NONE double-labeling mode and the Ct value is less than or equal to 36.0, judging that CSFV exists in the sample to be detected;

if the FAM/NONE double-labeling mode and the ROX/NONE double-labeling mode do not have a specific amplification curve, and the Ct value is larger than 39.0 or no Ct, determining that the PRRSV and CSFV do not exist in the sample to be detected;

if the FAM/NONE double-labeling mode and/or the ROX/NONE double-labeling mode have specific amplification curves, but the Ct value is more than 36.0 and less than or equal to 39.0, the test needs to be repeated.

The invention has the beneficial effects that:

the invention respectively uses ORF6 gene of PRRSV and 5' UTR non-coding region of CSFV as amplification target region, designs primer probe group aiming at PRRSV and primer probe group aiming at CSFV, and ensures the conservation and stability of detection gene locus. The primer probe group of the PRRSV and the primer probe group aiming at the CSFV have no interaction between the sequence structures, the amplification efficiency and the combination rate are high, the optimized reaction condition is suitable, the kit can be used for quantitative detection of trace PRRSV and CSFV, and has high detection sensitivity.

Based on the primer probe set provided by the invention, the fluorescent quantitative PCR technology is adopted to realize the rapid identification and detection of PRRSV and CSFV in one reaction, has the advantages of rapidness, specificity, sensitivity, high flux and the like, can meet the requirements of large-scale and rapid identification and detection of porcine reproductive and respiratory syndrome virus and classical swine fever virus, and provides an effective means for the detection and prevention and control of porcine reproductive and respiratory syndrome virus and classical swine fever virus.

The detection reagent kit containing the primer probe group aiming at the PRRSV and the primer probe group aiming at the CSFV, which is provided by the invention, is suitable for detecting virus nucleic acid in samples such as serum, spleen, lymph node, tonsil or kidney of suspected PRRSV or CSFV infected pigs, and the detection sensitivity can reach 23 copies/mu l. The virus has no cross reaction with other pathogens like mixed infection or infection symptoms of porcine reproductive and respiratory syndrome virus and classical swine fever virus, such as African Swine Fever Virus (ASFV), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), Porcine Parvovirus (PPV), porcine encephalitis B virus (JEV) and Haemophilus Parasuis (HPS).

Drawings

FIG. 1 is a PRRSV-sensitive amplification curve obtained in example 6;

figure 2 is a PRRSV standard curve obtained in example 6;

FIG. 3 is a CFSV sensitive amplification curve obtained in example 6;

FIG. 4 is a CFSV standard curve obtained in example 6;

FIG. 5 is an amplification curve obtained by the double fluorescent quantitative PCR detection method of PRRSV and CSFV in example 7.

Detailed Description

The technical solutions of the present invention are further described below with reference to the embodiments, but the present invention is not limited thereto, and any modifications or equivalent substitutions made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention should be covered by the protection scope of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are conventional in the art, and if not specifically mentioned, the raw materials and the like used in the examples of the present invention are commercially available; unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

Example 1

The invention provides a primer probe set for double fluorescent quantitative PCR detection of PRRSV and CSFV, which comprises a primer probe set aiming at PRRSV and a primer probe set aiming at CSFV.

Primer probe sets for PRRSV include primers and probes having the following nucleotide sequences:

forward primer PRRSV-D1-F: 5'-ACCTGGAAATTCATCACCTC-3', as shown in SEQ ID No. 1;

reverse primer PRRSV-D1-R: 5'-CGACAAATGCGTGGTTATCA-3', as shown in SEQ ID No. 2;

probe PRRSV-Probe: 5 '-FAM-TGCTAGGCCGCAAGTACATTC-BHQ 1-3' as shown in SEQ ID No. 3;

forward primer CSFV-D155-18F: 5'-GGGTGGTCTAAGTCCTGA-3', as shown in SEQ ID No. 4;

reverse primer CSFV-D325-18R: 5'-CTAATAGTGGGCCTCTGC-3', as shown in SEQ ID No. 5;

probe CSFV-Probe: 5 '-ROX-CAGTAGTTCGACGTGAGCAGAAG-BHQ 2-3' as shown in SEQ ID No. 6.

The primer probe set aiming at the PRRSV and the primer probe set aiming at the CSFV, which are designed by the invention, respectively take ORF6 gene of the PRRSV and 5' UTR non-coding region of the CSFV as amplification target regions, thereby ensuring the conservation and stability of detection gene sites. The sequence structures of the two groups of primer probe sets do not have interaction, the amplification efficiency and the binding rate are high, and the detection sensitivity is very high.

Example 2

The present embodiment provides a kit for dual fluorescent quantitative PCR detection of PRRSV and CSFV, comprising a primer probe set for PRRSV and a primer probe set for CSFV in embodiment 1: a forward primer PRRSV-D1-F, a reverse primer PRRSV-D1-R, a Probe PRRSV-Probe, a forward primer CSFV-D155-18F, a reverse primer CSFV-D325-18R and a Probe CSFV-Probe.

The kit also comprises DNA polymerase, reverse transcriptase mix, negative control, PRRSV and CSFV positive control and PCR buffer solution containing dNTP.

The PCR Buffer containing dNTP contains Buffer, magnesium chloride and dNTP, and the commercial 2 Xone Step RT-PCR Buffer III is used in the kit of the embodiment.

The DNA polymerase used in the kit of this example was TaKaRa Ex Taq HS, which is commercially available at 5U/. mu.l.

The reverse transcriptase cocktail contained RNase inhibitor, M-MuLV reverse transcriptase and thermostable DNA polymerase, and the commercial PrimeScript RT Enzyme Mix II was used in the kit of this example.

The negative control used in the kit of this example was sterilized diethyl pyrocarbonate-treated water, i.e., sterile DEPC-H₂O。

The PRRSV and CSFV positive reference substance used in the kit of this embodiment is a mixture of PRRSV virus positive plasmid and CSFV virus positive plasmid, and the concentrations of PRRSV virus positive plasmid and CSFV virus positive plasmid in the mixture are both 2.4 × 10^-4ng/ul。

The PRRSV virus positive plasmid is a recombinant plasmid constructed by connecting a product obtained by amplifying a target sequence by taking a nucleotide sequence shown as SEQ ID No.7 in a PRRSV ORF6 gene sequence conserved fragment and a vector, and a primer pair used for amplification has the following nucleotide sequence:

PRRSV-SF：5′-TTTCAGCGGAACAATGGGGTC-3′；

PRRSV-SR：5′-TTTCTGCCACCCAACACGAGG-3′；

the positive plasmid of the CSFV virus is a recombinant plasmid constructed by connecting a product obtained by amplifying a target sequence by taking a nucleotide sequence shown as SEQ ID No.10 in a conserved fragment of a CSFV 5' UTR gene sequence and a vector, wherein a primer pair used for amplification has the following nucleotide sequence:

CSFV-S149-18F；5′-CTCCCTGGGTGGTCTAAG-3′；

CSFV-S729-18R；5′-GCTACCTGTCACCCTACC-3′。

example 3

The embodiment provides a preparation method of PRRSV positive plasmid and positive standard substance, which comprises the following steps:

step one, obtaining PRRSV genome RNA:

the PRRSV is derived from a commercial vaccine TJM-F92 strain, and genomic RNA of the PRRSV is extracted from the commercial vaccine by using a full-automatic nucleic acid extractor HERO 32 (Loyang Aison) (magnetic bead method extraction reagent, Loyang Aison).

Step two, obtaining a target amplification product of PRRSV:

using Takara PrimeScript^TMOne Step reagent of One Step RT-PCR Kit Ver.2(RR055), genome RNA of PRRSV as template, PRRSV ORF6 gene sequence conserved fragment as amplification target sequence, and designed amplification primer with the following nucleotide sequence:

PRRSV-SF: 5'-TTTCAGCGGAACAATGGGGTC-3', as shown in SEQ ID No. 8;

PRRSV-SR: 5'-TTTCTGCCACCCAACACGAGG-3', as shown in SEQ ID No. 9;

and (2) performing conventional one-step PCR amplification by using the PRRSV genomic RNA obtained in the first step as a template and using amplification primers PRRSV-SF and PRRSV-SR, wherein an amplification system is shown in Table 2:

TABLE 2

Reagent	Amount of the composition used	Final concentration
			PrimeScript 1Step Enzyme Mix	2μl
2X 1Step Buffer	25μl
			PRRSV-SF(10μM)	2μl	0.4μM
PRRSV-SR(10μM)	2μl	0.4μM
			Genomic RNA	2μl
RNase Free dH2O	17μl

The amplification procedure is shown in table 3:

TABLE 3

Obtaining a target amplification product of PRRSV with the sequence shown as SEQ ID No. 7.

Step three, obtaining PRRSV positive plasmid:

and (3) connecting the target amplification product of the PRRSV obtained in the step (II) with a Vector pMD19-T (T-Vector pMD19 (Simple), Takara Code No.3271) to construct a recombinant plasmid, wherein the connecting and transforming method comprises the following steps:

1. the following reaction solutions (total volume 5. mu.l) were prepared in a microcentrifuge tube as shown in Table 4:

TABLE 4

T-Vector pMD19(Simple)	1μl
		Target amplification product of PRRSV	4μl

2. To the above DNA solution was added 5. mu.l of DNA Ligation Kit < Mighty Mix > (Takara Code number 6023), and mixed well.

The reaction was carried out at 3.16 ℃ for 30 min.

4. Mu.l of the reaction system was added to 100. mu.l of E.coli competent cells, gently mixed and then placed on ice for 30 min.

After being bathed in water at 5.42 ℃ for 45s, the mixture is quickly transferred into ice and placed for 1 min.

6. Adding 890. mu.l SOC culture medium, and culturing at 37 deg.C under shaking for 60 min.

7. Mu.l of the resulting transformation solution was applied to an L-agar plate medium containing X-Gal, IPTG and Amp, and cultured overnight at 37 ℃.

8. White colonies were picked and verified by PCR.

9. And (3) the positive recombinant plasmid with correct sequencing is the PRRSV positive plasmid.

Step four, obtaining a PRRSV positive standard substance:

amplifying the colony containing the PRRSV positive plasmid, extracting the PRRSV positive plasmid by using a commercial plasmid extraction kit to obtain a PRRSV positive standard substance, measuring OD260 and OD280 values and OD260/OD280 values of the PRRSV positive standard substance by using a spectrophotometer, and repeating for 5 times to obtain the PRRSV positive standard substance particle concentration of 242 ng/mul.

Reference plasmid DNA copy number calculation method: (6.02x10²³Hypocopies/mole) x (n)g/μl×10^-9) /(DNA length. times.660) ═ copies/. mu.l. Substituting into formula to calculate the concentration of DNA solution of PRRSV positive standard substance to be 7X 10¹⁰copies/. mu.l, stored at-20 ℃ until use.

Example 4

The embodiment provides a preparation method of a CSFV positive plasmid and a positive standard substance, which comprises the following steps:

step one, obtaining CSFV genome RNA:

CSFV is derived from a commercial subculture cell source, namely a swine fever live vaccine, and genomic RNA of PRRSV (magnetic bead extraction reagent, Loyang Aison) is extracted from the commercial vaccine by using a full-automatic nucleic acid extractor HERO 32 (Loyang Aison).

Step two, obtaining a target amplification product of CSFV:

using Takara PrimeScript^TMOne Step method reagent of One Step RT-PCR Kit Ver.2(RR055), genome RNA of CSFV is template, conserved fragment of CSFV 5' UTR gene sequence is used as target sequence of amplification, and an amplification primer with the following nucleotide sequence is designed:

CSFV-S149-18F; 5'-CTCCCTGGGTGGTCTAAG-3', as shown in SEQ ID No. 11;

CSFV-S729-18R; 5'-GCTACCTGTCACCCTACC-3', as shown in SEQ ID No. 12;

performing conventional PCR amplification by using the CSFV cDNA template obtained in the step one and the amplification primers CSFV-S149-18F and CSFV-S729-18R, wherein the amplification system is shown in Table 5:

TABLE 5

Reagent	Amount of the composition used	Final concentration
			PrimeScript 1Step Enzyme Mix	2μl
2X 1Step Buffer	25μl
			CSFV-S149-18F	2μl	0.4μM
CSFV-S729-18R	2μl	0.4μM
			Genomic RNA	2μl
RNase Free dH2O	17μl

The amplification procedure is shown in table 6:

TABLE 6

Obtaining a target amplification product of CSFV with the sequence shown as SEQ ID No. 10.

Step three, obtaining a CSFV positive plasmid:

connecting the target amplification product of CSFV obtained in the step two with a Vector pMD19-T (T-Vector pMD19 (Simple), Takara Code No.3271) to construct a recombinant plasmid, wherein the connecting and transforming method comprises the following steps:

1. the following reaction solutions (total volume 5. mu.l) were prepared in a microcentrifuge tube as shown in Table 7:

TABLE 7

T-Vector pMD19(Simple)	1μl
		Target amplification product of CSFV	4μl

The reaction was carried out at 3.16 ℃ for 30 min.

7. Mu.l of the transformation solution was applied to an L-agar plate medium containing X-Gal, IPTG and Amp, and cultured overnight at 37 ℃.

8. White colonies were picked and verified by PCR.

9. And (3) the positive recombinant plasmid with correct sequencing is the CSFV positive plasmid.

Step four, obtaining a CSFV positive standard substance:

amplifying the colony containing the CSFV positive plasmid, extracting the CSFV positive plasmid by using a commercial plasmid extraction kit to obtain a CSFV positive standard substance, measuring OD260 and OD280 values and OD260/OD280 values of the CSFV positive standard substance by using a spectrophotometer, and repeating for 5 times to obtain the CSFV positive standard substance with the plasmid concentration of 125 ng/mul.

Reference plasmid DNA copy number calculation method: (6.02x10²³Hypocopies/mole) x (ng/. mu.l.times.10^-9) /(DNA length. times.660) ═ copies/. mu.l. Substituting into formula to calculate the concentration of DNA solution of CSFV positive standard substance to be 3.5X 10¹⁰copies/. mu.l, stored at-20 ℃ until use.

Example 5

The present embodiment provides a method for detecting a sample to be detected by using the kit for dual fluorescent quantitative PCR detection of PRRSV and CSFV provided in embodiment 2, comprising the following steps:

(one) sample requirement:

the detection kit and the detection method provided by the embodiment are suitable for detecting the virus nucleic acid in the serum, spleen, lymph node, tonsil, kidney and other samples of suspected PRRSV and/or CSFV infected pigs.

Samples suspected of infecting pigs are collected by applying the conventional correct technology, and sediments and suspended matters which can influence the test result in the samples are removed by centrifugation. After being treated and collected, the sample is placed at room temperature for no more than 6 hours, and if the sample is not detected within 6 hours, the sample needs to be placed in a refrigerator at the temperature of 2-8 ℃ for storage; if the food needs to be stored or transported for more than 24 hours, the food should be frozen below-20 ℃ to avoid repeated freezing and melting. Before use, return to room temperature and mix by gentle shaking.

(II) obtaining a sample to be detected:

directly taking 100 mul of supernatant as a sample to be detected after the serum sample is centrifuged; taking about 0.5g of tissue samples such as spleen, lymph node, tonsil, kidney and the like, fully grinding, adding 0.3mL of PBS (pH7.4), fully mixing, centrifuging at 10000r/min for 8-10 min, and taking supernatant as a sample to be detected.

And (III) extracting the genome of the sample to be detected:

and (3) adopting a commercialized genome rapid extraction kit (centrifugal column type) product, and operating according to a kit specification to obtain the genome of the sample to be detected. Alternatively, a commercial full-automatic nucleic acid extractor HERO 32 (luoyang elsen) can be used, and the genome of the sample to be tested can be obtained by operating according to the instructions of instruments and kits (magnetic bead extraction reagent, luoyang elsen) of various manufacturers.

(IV) double fluorescent quantitative PCR amplification:

premixing a fluorescent quantitative PCR reaction system into a fluorescent PCR reaction tube, and preparing RT-PCR reaction liquid according to the following components (the preparation of the reaction liquid is carried out on ice). The Buffer and Enzyme Mix of the kit are One Step PrimeScript from Takara^TMRT-PCR Kit (Perfect Real Time) (Code No. RR064), the amplification system is shown in Table 8:

TABLE 8

Reagent	Amount of the composition used	Final concentration
			2X One Step RT-PCR BufferⅢ	10μl
TaKaRa Ex Taq HS(5U/μl)	0.4μl
			PrimeScript RT Enzyme MixⅡ	0.4μl
PRRSV-D1-F(10μM)	0.4μl	0.2μM
			PRRSV-D1-R(10μM)	0.4μl	0.2μM
CSFV-D155-18F(10μM)	0.4μl	0.2μM
			CSFV-D325-18R(10μM)	0.4μl	0.2μM
PRRSV-Probe	0.8μl	0.4μM
			CSFV-Probe	0.8μl	0.4μM
Amplification template	2μl
			RNase Free dH2O	4μl
Total	20μl

And respectively adding the genome of the sample to be detected, the PRRSV and the positive control substance and the negative control substance of the CSFV into different fluorescent PCR reaction tubes by taking the genome of the sample to be detected, the PRRSV and the CSFV as amplification templates, centrifuging all the fluorescent PCR reaction tubes at 8000r/min for several seconds, and putting the tubes into an RT-PCR amplification instrument.

The probe detection mode is set as follows:

the fluorescent mode PRRSV is FAM/NONE double-labeling mode, and CSFV is ROX/NONE double-labeling mode.

The double fluorescent quantitative PCR amplification reaction program is the first stage, reverse transcription is carried out at 42 ℃ for 5min, pre-denaturation is carried out at 95 ℃ for 10s, and 1 cycle is carried out; the second stage, 95 ℃ 5s, 60 ℃ 34s, for 40 cycles, and fluorescence signal detection was performed at the end of extension of each cycle.

And step two, analyzing the PCR product obtained by amplification in the step one, and judging whether the sample to be detected contains PRRSV and/or CSFV or not based on the specific amplification curve generation condition and the Ct value.

Quality control

Negative control: the detection results in the "FAM/NONE" and "ROX/NONE" labeling modes should be amplification curve log-increment free, with Ct values > 39.0 or no Ct.

Positive control: 2 amplification curves with obvious logarithmic growth period appear under the marking modes of FAM/NONE and ROX/NONE, and the Ct value is less than or equal to 25.0.

The above requirements need to be met simultaneously in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.

The second detection result judging method comprises the following steps:

if the FAM/NONE double-labeled mode and/or the ROX/NONE double-labeled mode have specific amplification curves, but the Ct value is more than 36.0 and less than or equal to 39.0, the test needs to be repeated.

The threshold value is set based on the condition that the threshold value line just exceeds the highest point of the amplification curve of the normal negative control substance, and can be adjusted according to the noise conditions of different instruments.

Example 6

The example verifies the sensitivity of the PRRSV and CSFV double fluorescence quantitative PCR detection method, and establishes a standard curve, and the specific verification method is as follows:

firstly, obtaining nucleic acid templates with different dilution ratios:

the concentration obtained in example 3 was taken to be 7X 10¹⁰DNA solution of copies/. mu.l PRRSV positive standard and concentration 3.5X 10 obtained in example 4¹⁰Respectively carrying out 10-fold gradient dilution on copies/mu l of DNA solution of the CSFV positive standard substance, mixing the PRRSV gradient dilution solution and the CSFV gradient dilution solution with the same dilution ratio according to the volume ratio of 1:2, and obtaining the concentration of PRRSV and CSFV positive plasmid in the mixed solution under each dilution ratio which is 2.33 multiplied by 10 in turn⁶、2.33× 10⁵、2.33×10⁴、2.33×10³、2.33×10²And 2.33X 10¹copies/μl。

And secondly, performing RT-PCR by taking sterile deionized water as a negative control and mixed liquor under each dilution multiple as a nucleic acid template according to the detection method of the embodiment 5 to obtain a PRRSV sensitive amplification curve shown in the figure 1 and Ct values corresponding to the initial concentrations of different PRRSV positive plasmids shown in the table 9.

TABLE 9

The concentration of the PRRSV positive recombinant plasmid initial template is taken as an X axis, the Ct value of the RT-PCR cycle times is taken as a Y axis to be taken as a regression curve, a standard curve for detecting the PRRSV by the PRRSV and CSFV double RT-PCR detection method is established, and the result is shown in figure 2. Establishing PRRSV standard curve and correlation coefficient R according to sensitivity detection result²0.99, slope-3.536, and intercept 38.027, giving a linear relationship expression between copy number (X) and Ct value (y):

y＝-3.536logX+38.027。

sensitivity verification results show that the primers and the probes designed by the invention have high amplification efficiency and binding rate, the optimized reaction conditions are appropriate, PRRSV ORF6 gene positive control RNA molecules with the concentration of at least 23 copies/mu l can be detected, the primers and the probes can be used for quantitative detection of trace PRRSV, and the primers and the probes have very high detection sensitivity.

And thirdly, performing RT-PCR by using sterile deionized water as a negative control and mixed liquor under each dilution multiple as a nucleic acid template according to the detection method of the embodiment 5 to obtain a CFSV sensitive amplification curve shown in the figure 3 and Ct values corresponding to the initial concentrations of different CFSV positive plasmids shown in the table 10.

Watch 10

The initial template concentration of the CSFV positive recombinant plasmid is taken as an X axis, the Ct value of the RT-PCR cycle times is taken as a Y axis to be used as a regression curve, and a standard curve for detecting CSFV by a PRRSV and CSFV double RT-PCR detection method is established, and the result is shown in figure 4. Establishing a CSFV standard curve and a correlation coefficient R according to the sensitivity detection result²0.99, slope-3.371, and intercept 39.394, giving a linear relationship expression between copy number (X) and Ct value (y):

y＝-3.371logX+39.394。

sensitivity verification results show that the primers and the probes designed by the invention have high amplification efficiency and binding rate, the optimized reaction conditions are appropriate, and the primers and the probes can detect CSFV 5 'UTR gene positive control RNA molecules with the concentration of at least 23 copies/mu l, can be used for quantitative detection of trace CSFV 5' UTR genes, and have high detection sensitivity.

Example 7

This example demonstrates the specificity of the PRRSV and CSFV duplex fluorescent quantitative PCR detection method.

Extracting nucleic acid from commercial vaccines of African swine fever ASFV, porcine pseudorabies virus PRV, porcine circovirus 2 type PCV2, porcine parvovirus PPV, porcine Japanese encephalitis virus JEV and Haemophilus parasuis HPS by using a full-automatic nucleic acid extractor, performing reverse transcription to respectively obtain a virus nucleic acid sample to be detected, performing RT-PCR by using the kit of example 2 and the detection method of example 5 with PRRSV positive standard and CSFV positive standard as positive controls and sterile deionized water as negative controls, and obtaining a result shown in FIG. 5, wherein 1 is a CSFV positive standard amplification curve; 2 is PRRSV positive standard substance amplification curve; 3 is a negative control amplification curve; 4-9 are ASFV, PRV, PCV2, PPV, JEV, HPS amplification curves.

The Ct value of the RT-PCR amplification of the PRRSV positive standard substance is 18.739, the Ct value of the RT-PCR amplification of the CSFV positive standard substance is 22.8, and a specific amplification curve appears. No specific amplification curve appears in ASFV, PRV, PCV2, PPV, JEV, HPS, negative control and the like. This demonstrates that the primer probe set for PRRSV and the primer probe set for CSFV provided by the present invention do not cross react with other pathogens that are susceptible to mixed infection or symptoms of infection similar thereto, and have significant specificity.

Example 8

This example demonstrates the stability and reproducibility of the PRRSV and CSFV duplex fluorescence quantitative PCR detection method.

The concentration obtained in example 3 was taken to be 7X 10¹⁰DNA solution of copies/. mu.l PRRSV positive standard and concentration 3.5X 10 obtained in example 4¹⁰Respectively diluting the DNA solution of the copies/mu l of CSFV positive standard substance by 10 times of gradient, and diluting the PRRSV gradient dilution solution and the CSFV gradient dilution with the same dilution timesThe diluted liquid is mixed according to the volume ratio of 1:2, and the concentration of PRRSV and CSFV positive plasmid in the mixed liquid under 4 dilution times is 2.33 multiplied by 10 in sequence⁵、 2.33×10⁴、2.33×10³And 2.33X 10²copies/μl。

RT-PCR was performed according to the detection method of example 5 using sterile deionized water as a negative control and the mixture at each dilution as a nucleic acid template, and 3 replicates were set for each series to obtain the test results shown in tables 11 and 12.

TABLE 11

TABLE 12

The results in tables 11 and 12 show that the variation coefficient (CV value) of the PRRSV and CSFV intra-batch repeat and inter-batch repeat tests is below 1 percent, and the PRRSV and CSFV double RT-PCR detection method established by the invention has good stability and repeatability.

SEQUENCE LISTING

<110> Harbin Zhongke Gene technology Co., Ltd

<120> primer probe set, kit and detection method for PRRSV and CSFV double fluorescence quantitative PCR detection

<130> 1

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

acctggaaat tcatcacctc 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

cgacaaatgc gtggttatca 20

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<400> 3

tgctaggccg caagtacatt c 21

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

<400> 4

gggtggtcta agtcctga 18

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence

<400> 5

ctaatagtgg gcctctgc 18

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<400> 6

cagtagttcg acgtgagcag aag 23

<210> 7

<211> 492

<212> DNA

<213> Porcine Reproductive and Respiratory Syndrome Virus

<400> 7

tttcagcgga acaatggggt cgtctctaga cgacttttgc catgatagca cggctccaca 60

aaaggtgctt ttggcgtttt ccattaccta tacgccagtg atgatatatg ctctaaaggt 120

aagtcgcggc cgactgctag ggcttctgca ccttttgatc tttctgaatt gttcttttac 180

cttcgggtac atgacattcg tgcacttcca gagcacaaat agggtcgcgc tcactatggg 240

agcagtagtt gcacttcttt ggggagtgta ctcggccata gaaacctgga aattcatcac 300

ctccagatgc cgtttgtgct tgctaggccg caagtacatt ctggcccctg cccaccacgt 360

cgaaagtgcc gcgggctttc atccgattgc ggcaaatgat aaccacgcat ttgtcgtccg 420

gcgtcccggc tccactacgg ttaacggcac attggtgccc gggttgaaag gcctcgtgtt 480

gggtggcaga aa 492

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

tttcagcgga acaatggggt c 21

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<400> 9

tttctgccac ccaacacgag g 21

<210> 10

<211> 597

<212> DNA

<213> Classical Swine Fever Virus

<400> 10

ctccctgggt ggtctaagtc ctgagtacag gacagtcgtc agtagttcga cgtgagcaga 60

agcccacctc gagatgctat gtggacgagg gcatgcccaa gacacacctt aacctagcgg 120

gggtcgttag ggtgaaatca caccatgtga tgggagtacg acctgatagg gtgctgcaga 180

ggcccactat taggctagta taaaaatctc tgctgtacat ggcacatgga gttgaatcat 240

tttgaacttt tatacaaaac aaacaaacaa aaaccaatgg gagtggagga accggtatac 300

gatgtaacgg ggagaccatt gtttggagac ccaagtgagg tacacccaca atcaacattg 360

aagctaccac atgatagggg gagaggcaac atcaaaacaa cactgaagaa tctacctagg 420

agaggtgact gcaggagtgg caaccaccta ggcccggtta gtgggatata tgtaaagccc 480

ggccctgtct tttatcagga ctacatgggc ccagtctatc atagagcccc tctggagttt 540

tttgacgaag cacagttctg tgaggtgacc aaaaggatag gtagggtgac aggtagt 597

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<400> 11

ctccctgggt ggtctaag 18

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence

<400> 12

gctacctgtc accctacc 18

Claims

1. A primer probe group for double fluorescent quantitative PCR detection of PRRSV and CSFV is characterized by comprising a primer probe group aiming at PRRSV and a primer probe group aiming at CSFV;

forward primer PRRSV-D1-F: 5'-ACCTGGAAATTCATCACCTC-3', respectively;

reverse primer PRRSV-D1-R: 5'-CGACAAATGCGTGGTTATCA-3', respectively;

probe PRRSV-Probe: 5 '-FAM-TGCTAGGCCGCAAGTACATTC-BHQ 1-3';

forward primer CSFV-D155-18F: 5'-GGGTGGTCTAAGTCCTGA-3', respectively;

reverse primer CSFV-D325-18R: 5'-CTAATAGTGGGCCTCTGC-3', respectively;

probe CSFV-Probe: 5 '-ROX-CAGTAGTTCGACGTGAGCAGAAG-BHQ 2-3'.

2. A kit for double fluorescent quantitative PCR detection of PRRSV and CSFV, which is characterized in that the kit comprises the forward primer PRRSV-D1-F, the reverse primer PRRSV-D1-R, the Probe PRRSV-Probe, the forward primer CSFV-D155-18F, the reverse primer CSFV-D325-18R and the Probe CSFV-Probe of claim 1.

3. The kit for the double fluorescent quantitative PCR detection of PRRSV and CSFV according to claim 2, characterized in that it further comprises DNA polymerase, reverse transcriptase mix, negative control, PRRSV and CSFV positive control and PCR buffer containing dNTP.

4. The kit for dual fluorescent quantitative PCR detection of PRRSV and CSFV according to claim 3, characterized in that the positive control of PRRSV and CSFV is a mixture of PRRSV virus positive plasmid and CSFV virus positive plasmid, and the concentration of PRRSV virus positive plasmid and CSFV virus positive plasmid in the mixture is 2.4 x10^-4ng/ul; the negative control substance is sterile DEPC-H₂O。

5. The kit for the double fluorescent quantitative PCR detection of PRRSV and CSFV according to claim 4, characterized in that the PRRSV positive plasmid is a recombinant plasmid constructed by connecting a product obtained by amplifying a target sequence with a nucleotide sequence shown as SEQ ID No.7 in a PRRSV ORF6 gene sequence conserved fragment and a vector, and a primer pair used for amplification has the following nucleotide sequence:

PRRSV-SF：5′-TTTCAGCGGAACAATGGGGTC-3′；

PRRSV-SR：5′-TTTCTGCCACCCAACACGAGG-3′；

the positive plasmid of the CSFV virus is a recombinant plasmid constructed by connecting a product obtained by amplifying a target sequence by taking a nucleotide sequence shown as SEQ ID No.10 in a CSFV 5' UTR gene sequence conserved fragment and a vector, and a primer pair used for amplification has the following nucleotide sequence:

CSFV-S149-18F；5′-CTCCCTGGGTGGTCTAAG-3′；

CSFV-S729-18R；5′-GCTACCTGTCACCCTACC-3′。

6. a method for double fluorescent quantitative PCR detection of PRRSV and CSFV is characterized by comprising the following steps:

7. The method of claim 6, wherein the double RT-PCR amplification reaction system of step one comprises the forward primer PRRSV-D1-F, the reverse primer PRRSV-D1-R, the forward primer CSFV-D155-18F and the reverse primer CSFV-D325-18R with final concentration of 0.2 μ M, the Probe PRRSV-Probe and the Probe CSFV-Probe with final concentration of 0.4 μ M, the PCR buffer solution 10 μ l, the DNA polymerase 5U/μ l 0.4 μ l, the reverse transcriptase cocktail 0.4 μ l, the amplification template 2 μ l, the RNase Free dH₂O make up to a total volume of 20. mu.l.

8. The method of claim 7, wherein the dual RT-PCR amplification reaction procedure of step one is a first stage: reverse transcription at 42 deg.C for 5min, pre-denaturation at 95 deg.C for 10s, and 1 cycle; and a second stage: 5s at 95 ℃ and 34s at 60 ℃ for 40 cycles.

9. The method of claim 8, wherein the probe detection mode of the step-one fluorescent signal detection is set as follows: the fluorescent mode PRRSV is FAM/NONE double-labeling mode, and CSFV is ROX/NONE double-labeling mode.

10. The method for the double fluorescent quantitative PCR detection of PRRSV and CSFV according to claim 9, characterized in that the determination method of the detection result in the second step is:

if a specific amplification curve appears only in the FAM/NONE double-labeling mode and the Ct value is less than or equal to 36.0, determining that PRRSV exists in the sample to be detected;