CN106947834B - Multiplex PCR method for detecting six duck susceptibility viruses - Google Patents

Abstract

The invention discloses a multiplex PCR method for detecting six duck susceptibility viruses, which comprises the following steps: preparing a genome; designing and synthesizing a primer; detecting the specificity of the primer; establishing a multiplex PCR system; multiplex PCR specific detection. The invention establishes an m-PCR method for rapidly identifying and diagnosing 6 common viruses of ducks in a single reaction system, and is reported for the first time at home and abroad. The method has the advantages of short detection time, strong specificity, good accuracy and higher sensitivity, and can be widely applied to the rapid differential diagnosis of the duck virus infection sample in clinic.

Description

Multiplex PCR method for detecting six duck susceptibility viruses

Technical Field

The invention belongs to the technical field of epidemiology, and particularly relates to a multiplex PCR (polymerase chain reaction) method for detecting six duck susceptibility viruses.

Background

Viral infectious diseases are important diseases which harm duck groups. In recent years, various factors such as scale enlargement of duck breeding industry, increase of mixed breeding modes, enhancement of mobility of people and livestock, water quality reduction caused by water source pollution and the like create favorable conditions for virus propagation, local epidemic viral diseases are obviously increased, new epidemic situations are continuously generated, and serious economic loss is caused to the duck breeding industry. At present, main viruses for infecting ducks comprise duck hepatitis virus type 1, duck plague virus, duck tembusu virus, Muscovy duck parvovirus, Muscovy duck reovirus, duck H9N2 low-pathogenicity avian influenza virus and the like, a rapid detection method which can be generally applicable to the infection of the viruses is established, prevention and treatment measures are taken at present, and the reduction of economic loss is an important problem which troubles the duck breeding industry.

Clinically, the technologies of virus isolation and identification, serological detection, immune electron microscopy, ELISA, Real-time PCR, LAMP, common PCR and the like are respectively applied to the detection of virus infection, but the virus isolation and identification are long in time consumption, the serological and ELISA detection are time-consuming and labor-consuming, the immune electron microscopy and Real-time PCR have high requirements on equipment and virus purification, and the limitations are brought to clinical diagnosis. The PCR detection technology has high sensitivity, is simple, convenient, rapid, safe and easy to popularize, can detect without separating viruses, and has been reported to be applied to the detection of duck hepatitis viruses, reoviruses, H7 subtype influenza viruses, Muscovy duck parvoviruses and duck plague viruses, but can not be used for identifying and diagnosing other viruses. Multiplex PCR (multiplex xPCR, m-PCR) is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments, and the process and principle are the same as those of ordinary PCR, but the multiplex PCR has incomparable advantages. In the aspects of viral disease detection and differential diagnosis, the m-PCR can simultaneously detect and identify a plurality of viruses, and is an effective method for clinically and rapidly diagnosing virus mixed infection. At present, no report of m-PCR of common viral diseases of ducks at home and abroad exists,

disclosure of Invention

The invention aims to provide a multiplex PCR method for detecting six duck susceptibility viruses. By comparing related gene sequences of different virus proteins and designing a specific primer without cross amplification according to a conserved region sequence, an m-PCR method which is generally suitable for rapidly detecting a duck virus infection sample in clinic is established.

The specific technical scheme is as follows:

a multiplex PCR method for detecting six duck susceptibility viruses comprises the following steps:

step 1, genome preparation

Respectively extracting genome RNA/DNA of existing strains of 6 viruses according to the instruction of a Viral RNA/DNA Extraction Kit, and measuring the concentration and the purity by a spectrophotometer after the nuclease-free ultrapure water is dissolved; the RNA of DHAV-1, DTMUV, ARV and H9N2 is reversely transcribed into cDNA according to the MMLV specification, and all the DNA/cDNA is stored at-80 ℃ for later use;

step 2, primer design and synthesis

Respectively comparing protein sequences of 6 viruses such as DHAV-1, DPV, DTMUV, MPDV, ARV and H9N2 published in Genebank with virus protein sequences of strains existing in laboratories, selecting conserved regions, and utilizing Primer Premier V5.0 and Oligo V6.22 software to evaluate and screen primers, wherein specific primers are designed as shown in SEQ ID NO 1-12, and the primers are synthesized by Yinfei Jieji (Shanghai) trade Limited;

step 3, detecting the specificity of the primer

Respectively taking DNA/cDNA of existing strains of 6 viruses as templates, amplifying corresponding target fragments, wherein the total volume of each reaction system is 50 mu l, and the reaction system comprises 5 mu l of single virus template, 1 mu l of respective upstream and downstream primers, 25 mu l of 2 XTaqmix enzyme and 18 mu l of water; the reaction procedure is as follows: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing at 52 deg.C for 45s, and extension at 72 deg.C for 1min, performing 30 cycles, and finally extension at 72 deg.C for 10 min; after the reaction is finished, 5 mul of product is taken to carry out 1.5 percent agarose gel electrophoresis analysis, the product is sent to Weijie Weiji base (Shanghai) trade company Limited for sequencing after being purified, and the accuracy of the amplified product is verified; meanwhile, a blank control group without DNA is arranged;

carrying out PCR amplification by taking DNA/cDNA mixtures of existing strains of 6 viruses as templates, wherein each reaction system is 50 mu l, and comprises 23 mu l (enough amount) of DNA/cDNA mixture, 1 mu l of upstream and downstream primers of the viruses and 25 mu l of 2 XTaqmix enzyme respectively, and the reaction program, the electrophoresis and the like are the same as above;

step 4, establishing a multiple PCR system

Mixing 6 pairs of PCR primers, using 6 strains of virus DNA/cDNA mixture (sufficient amount) as template, 50. mu.l reaction system, 0.2. mu.M primer final concentration, 52 ℃ annealing temperature, 25 cycles, and analyzing PCR product by 1.5% agarose gel electrophoresis, and comparing with expected fragment size;

step 5, multiplex PCR specificity detection

According to the established multiplex PCR method, PCR amplification is carried out by taking DNA/cDNA of each virus strain as a template, wherein each reaction system is 50 mu l, and comprises 5 mu l of single virus template, 12 mu l of 6 pairs of primer mixture, 25 mu l of 2 xTaqmix enzyme and 8 mu l of deionized water, and the reaction procedures are as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 45s, extension at 72 ℃ for 1min, performing 25 cycles, finally extension at 72 ℃ for 7min, and analyzing PCR products by 1.5% agarose gel electrophoresis; while deionized water was set as a blank control.

Compared with the prior art, the invention has the beneficial effects that:

the invention establishes an m-PCR method for rapidly identifying and diagnosing 6 common viruses of ducks in a single reaction system, and is reported for the first time at home and abroad. The method has the advantages of short detection time, strong specificity, good accuracy and higher sensitivity, and can be widely applied to the rapid differential diagnosis of the duck virus infection sample in clinic.

Drawings

FIG. 1 is an electrophoretogram of single viral PCR products amplified with a single primer, wherein,

1. blank control; 2. PCR products of each virus; m. DNA molecular weight Marker

FIG. 2 is an electrophoretogram of single primer amplified multiple viral PCR products, wherein,

m. DNA molecular weight Marker;

a multi-viral PCR product amplified with DPV primers;

multiple virus PCR products amplified with ARV primers;

3, multi-virus PCR products amplified by MPDV primers;

a multi-virus PCR product amplified by the DHAV-1 primer;

a multi-viral PCR product amplified by the DTMUV primer;

the multi-viral PCR product amplified with the H9N2 primer.

FIG. 3 is an electrophoretogram of multiplex PCR products, wherein,

1. a multiplex PCR product;

and M, DNA molecular weight Marker.

FIG. 4 shows the result of multiplex PCR-specific detection, in which,

1. blank control;

1. detecting DPV by multiplex PCR;

3. detecting MPDV by multiplex PCR;

4. detecting DHAV-1 by multiplex PCR;

5. detecting DTMUV by multiplex PCR;

6. multiplex PCR detection H9N 2.

2. Detecting ARV by multiplex PCR;

and M, DNA molecular weight Marker.

FIG. 5 shows the results of sensitivity testing of multiplex PCR, wherein FIG. 5-1 shows the sensitivity testing of multiplex PCR to a single template

1-7 multiplex PCR detection 10⁰～10⁶copies/. mu.l of diluted single template for each virus; m.100bp DNAladder; FIG. 5-2 shows the sensitivity of multiplex PCR to mixed templates, 1-8, multiplex PCR detection 10^-1～10⁶copies/μ l diluted 6 virus mixed template; m.100bp DNA ladder;

FIG. 6. simulation of multiple PCR detection results of mixed infection

M.100bp DNA ladder；

1-5, carrying out multiplex PCR detection on a simulated mixed infection sample;

6. a positive control;

7. blank control.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Materials and methods

1.1 Strain and Virus sample Collection

Duck hepatitis virus type 1 (DHAV-1-SH strain), duck plague virus (DPV-F34 strain), duck tembusu virus (DTMUV-JS804 strain), Muscovy duck parvovirus (MPDV-FJM3 strain) are gifted by Shanghai veterinary research institute of Chinese academy of agricultural sciences, Muscovy duck reovirus (ARV-HNF strain) are gifted by agricultural academy of Fujian province, and duck H9N2 low pathogenic avian influenza virus (H9N2-C/SH/F/98 strain) are gifted by veterinary academy of Yangzhou university; SPF duck embryos were purchased from harbin veterinary institute; the ARV-HNF strain is inoculated with 6-day-old duck embryos by yolk sac for amplification, and the DHAV-1-SH strain, the DTMUV-JS804 strain, the DPV-F34 strain, the H9N2-C/SH/F/98 strain and the MPDV-FJM3 strain are inoculated with 9-day-old duck embryos by allantoic cavities for amplification; clinical samples were taken from diseased ducks in duck farms in Jiangzhe area.

1.2 reagents and instruments

dNTPs, RNase inhibitor, Viral RNA/DNA Extraction Kit from TaKaRa (Dalian, China); 2 × Taqmix and DNA marker were purchased from Beijing kang, a century Biotechnology Co., Ltd; plasmid extraction kit, MMLV reverse transcription kit purchased from BBI company (USA); hydrochloride buffer, syringe, pipette, finger tube and paraffin were all commercially available. PCR instruments and cryo-centrifuges were purchased from Eppendorf; spectrophotometer was purchased from BIO-RAD; electrophoresis apparatus and full-automatic gel imaging processing system were purchased from Shanghai Tianneng technology, Inc.

1.3 genome preparation

Extracting genome RNA/DNA of existing strains of 6 viruses respectively according to the instruction of a Viral RNA/DNA Extraction Kit, and measuring the concentration and the purity by a spectrophotometer after the nuclease-free ultrapure water is dissolved. The RNA of DHAV-1, DTMUV, ARV and H9N2 was reverse transcribed into cDNA according to the MMLV protocol, and all DNA/cDNA was stored at-80 ℃ until use.

1.4 primer design and Synthesis

Protein sequences of 6 viruses DHAV-1, DPV, DTMUV, MPDV, ARV and H9N2 published in Genebank are respectively compared with virus protein sequences of strains existing in laboratories, conserved regions are selected, Primer Premier V5.0 and Oligo V6.22 software are used for evaluating and screening primers, specific primers are designed as shown in Table 1, and the primers are synthesized by Yiwei Jie (Shanghai) trade company Limited.

TABLE 1 primer sequence information

Note: F. an upstream primer; r. downstream primer.

1.5 primer specificity detection

The DNA/cDNA of the existing strains of 6 viruses are respectively used as templates to amplify corresponding target fragments, the total volume of each reaction system is 50 mu l, and the reaction system comprises 5 mu l of single virus template, 1 mu l of respective upstream and downstream primers, 25 mu l of 2 XTaqmix enzyme and 18 mu l of water. The reaction procedure is as follows: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing at 52 deg.C for 45s, and extension at 72 deg.C for 1min, performing 30 cycles, and finally extension at 72 deg.C for 10 min. After the reaction is finished, 5 mu l of product is taken to carry out 1.5 percent agarose gel electrophoresis analysis, the purified product is respectively cloned to pMD19-T vectors which are respectively named as pMD-DHAV-1, pMD-DPV, pMD-DTMUV, pMD-MPDV, pMD-ARV and pMD-H9N2, and the products are sent to Weijie Elegand (Shanghai) trade company Limited for sequencing, and the accuracy of the amplified product is verified. A blank control group without DNA was also set.

PCR amplification is carried out by taking DNA/cDNA mixtures of existing strains of 6 viruses as templates, wherein each reaction system is 50 mu l, 23 mu l (enough amount) of the DNA/cDNA mixture, 1 mu l of each of upstream and downstream primers of the viruses and 25 mu l of 2 XTaqmix enzyme are included, and the reaction program, the electrophoresis and the like are the same.

1.6 multiplex PCR System establishment

6 pairs of PCR primers were mixed, 6 strains of viral DNA/cDNA mixture (sufficient) were used as templates, conditions were re-optimized according to the established single PCR system and procedure, respectively setting 25, 50, 100. mu.l different reaction systems, 0.1, 0.2, 0.5. mu.M primer set final concentrations, 48, 50, 52, 54 ℃ annealing temperature, 15, 20, 25, 30 cycles, etc., and PCR products were analyzed by 1.5% agarose gel electrophoresis and compared with the expected fragment sizes.

1.7 multiplex PCR specific detection

According to the established multiplex PCR method, PCR amplification is carried out by taking DNA/cDNA of each virus strain as a template, wherein each reaction system is 50 mu l, and comprises 5 mu l of single virus template, 12 mu l of 6 pairs of primer mixture, 25 mu l of 2 xTaqmix enzyme and 8 mu l of deionized water, and the reaction procedures are as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 45s, extension at 72 ℃ for 1min, performing 25 cycles, and finally extension at 72 ℃ for 7min, and analyzing the PCR product by 1.5% agarose gel electrophoresis. While deionized water was set as a blank control.

1.8 multiplex PCR sensitivity evaluation

The recombinant plasmids corresponding to 6 virus genes are extracted by a plasmid extraction kit, wherein the recombinant plasmids are transcribed into ssRNA in vitro by a method of 4 plasmid references, namely pMD-DHAV-1, pMD-DTMUV, pMD-ARV and pMD-H9N 2. All ssRNA and plasmids pMD-MPDV, pMD-DPV were calibrated to 10 by spectrophotometer⁶Sensitivity of multiplex PCR to single template was determined by copies/μ l, 10-fold gradient dilution; mixing 6 single templates in equal concentrationFor multiple templates, the final concentration of each template was adjusted to 10⁶copies/. mu.l, the sensitivity of multiplex PCR to multiplex template was determined. The results were analyzed by observation on 1.5% agarose gel electrophoresis.

1.9 preliminary application of multiplex PCR

1.9.1 simulating mixed infection

Mixing virus containing 6 virus strains (each strain has an EID of 100)₅₀) Inoculating 5 SPF duck embryos of 10 days old through an allantoic cavity, respectively collecting embryo bodies and allantoic fluid after 72 hours, carrying out sterile shearing and grinding, adding a proper amount of PBS (phosphate buffer solution) to prepare homogenate, repeatedly freezing and thawing for 3 times, carrying out 5000r/min, centrifuging for 10min, and sucking supernatant liquid as a mixed infection sample. The total genome of the sample is extracted by a method of 1.3, the amplification is carried out by applying the established PCR method after the reverse transcription, and the amplification product is analyzed by 1.5 percent agarose gel electrophoresis. The duck embryo without virus infection is used as a blank control, and the mixed solution of 6 viruses is used as a positive control.

1.9.2 clinical sample testing

Collecting 87 parts of viscera mixture of suspected virus infected ducks in Jiangzhe area, aseptically shearing and grinding the viscera mixture, adding a proper amount of PBS (phosphate buffer solution) to prepare homogenate, preparing a detection sample according to a 1.9.1 method, extracting total DNA/RNA (deoxyribonucleic acid/ribonucleic acid) according to a 1.3 method, performing amplification by applying an established multiplex or single PCR (polymerase chain reaction) method after reverse transcription, and analyzing an amplification product by electrophoresis of 1.5% agarose gel. The single PCR amplification product with positive amplification result is sent to Weiji (Shanghai) trade company Limited for sequencing identification. The group of non-tissue samples was set as a blank control.

2. Results

2.1 primer specificity detection

Single DNA/cDNA of DHAV-1-SH strain, DPV-F34 strain, DTMUV-JS804 strain, MPDV-FJM3 strain, ARV-HNF strain and H9N2-C/SH/F/98 strain are used as templates, a single primer pair PCR amplifies target genes, 6 strains can specifically amplify fragments with expected sizes (shown in figure 1), negative control is not amplified, and a PCR sequencing result shows that a specific gene sequence is completely consistent with a target gene sequence. The results of single primer amplification of the viral DNA/cDNA mixture are shown in FIG. 2, where each primer pair amplifies only one specific band expected, and no non-specific amplification is observed.

2.2 multiplex PCR System establishment

Multiple PCR conditions are optimized on the basis of a single PCR system and a program, and a 50-microliter reaction system is found to be most easy to operate, the optimal final concentration of a primer is 0.2 microlitre, an expected band of any virus can be amplified at the annealing temperature of 52 ℃, non-specific amplification is avoided, each band can be clearly seen when the cycle is 25 times, and other conditions are the same as single PCR. Electrophoretic analysis revealed 6 clear bands that completely matched the expected fragment size (FIG. 3).

2.3 multiplex PCR specific detection

According to the established multiplex PCR method, single strain DNA/cDNA is amplified by 6 pairs of primers respectively, the result is shown in figure 4, each pair of primers is only combined with a corresponding template, and a target fragment with a corresponding size is amplified without a hybrid band. None of the bands were observed in the blank control group.

2.4 multiplex PCR sensitivity evaluation

The sensitivity of the established multiplex PCR method was determined and the results showed that the lowest detection values for DHAV-1, DPV, DTMUV, MPDV, ARV and H9N2 were all 10 copies/. mu.l when either ssRNA or plasmid DNA was used as the single template (FIG. 5-1). The lowest detection values for DHAV-1, DPV, MPDV and ARV were 10 when a mixed template of ssRNA and plasmid DNA was used²copies/. mu.l, the lowest detection of DTMUV and H9N2 was 10 copies/. mu.l (FIG. 5-2).

2.5 simulated Mixed infection test

According to the established multiplex PCR method, 5 SPF duck embryo samples infected by 6 virus strain mixed liquor are detected, the result is shown in figure 6, each sample can detect 6 specific bands with the same size as the positive control, and a blank control group does not amplify.

2.6 clinical sample testing

Detecting clinical samples by single or multiple PCR, wherein the positive amplification products of the single PCR are proved by sequencing identification that the positive results are all target fragments of corresponding viruses, 53 parts of 87 clinical samples are independently infected, wherein 13 parts of DHAV-1 infection, 9 parts of DPV infection, 10 parts of DTMUV infection, 3 parts of MPDV infection, 6 parts of ARV infection and 12 parts of H9N2 infection; 32 parts of multiple infection, 7 parts of DHAV-1+ DPV co-infection, 5 parts of DHAV-1+ DTMUV co-infection, 2 parts of ARV + H9N2 co-infection, 5 parts of H9N2+ DHAV-1 co-infection, 1 part of H9N2+ DTMUV co-infection, 6 parts of H9N2+ DPV co-infection, 1 part of MPDV + DPV infection, 1 part of DPV + DHAV-1+ DTMUV co-infection, 2 parts of H9N2+ ARV + DPV co-infection and 2 parts of H9N2+ DPV + DHAV-1 co-infection. The detection result of the multiplex PCR method is identical with the detection result of the single PCR.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims (1)

1. A primer for detecting six duck susceptibility viruses, wherein the six duck susceptibility viruses comprise DHAV-1, DPV, DTMUV, MPDV, ARV and H9N2, and the primer is characterized in that: the primer sequences are respectively as follows:

the primer sequence for detecting DHAV-1 virus is as follows:

F:GTTTGGGAGGCAATGGTT

R:ATTGAGTCCACATGAACAG；

the primer sequence for detecting the DPV virus is as follows:

F:GCCAGTGGACAGTTTGA

R:GGCCAGTTCTCCATTTG；

the primer sequence for detecting the DTMUV virus is as follows:

F:GGCTTGTTTGGAAAAGG

R:TTCCAGAGTACTTCACTG；

the primer sequence for detecting MPDV virus is as follows:

F:GGCATTGCGATTCCCAAT

R:GAGTCTCTGCCAGTCTC；

the primer sequence for detecting the ARV virus is as follows:

F:GCACTCTGGATCCAGTAC

R:CAATGGAGAAGCGAACCG；

the primer sequence for detecting the H9N2 virus is as follows:

F:CAAACTCCACAGAAACTG

R:CTGACATTGTGGAATGGC。

Images