Establishment and Application of a Multiplex PCR Method for the Simultaneous Detection of PRV, PCV2, PRRSV, CSFV, and SIV Associated with Porcine Respiratory Disease

doi:10.21203/rs.3.rs-3130329/v1

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Establishment and Application of a Multiplex PCR Method for the Simultaneous Detection of PRV, PCV2, PRRSV, CSFV, and SIV Associated with Porcine Respiratory Disease

https://doi.org/10.21203/rs.3.rs-3130329/v1

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Based on gene sequences in GenBank, five pairs of specific primers were designed to PCR amplify partial fragments of the genes encoding Pseudorabies virus (PRV) gB, Porcine circovirus type 2 (PCV2) ORF2, Porcine reproductive and respiratory syndrome virus(PRRSV) ORF6, Classical swine fever virus (CSFV) C, and Swine influenza virus (SIV) M. The fragments amplified were 192 bp (PRV), 255 bp (PCV2), 364 bp (PRRSV), 530 bp (CSFV), and 981bp (SIV). The results of sensitivity and specificity tests showed that the minimum amounts of nucleic acid required for the mPCR were 18 pg (PRV), 22 pg (PCV2), 26 pg (PRRSV), 48 pg (CSFV), and 32 pg (SIV). The amplification results for Porcine parvovirus and Porcine epidemic diarrhea virus were both negative. The results for 48 clinical samples collected in Guizhou Province revealed that the five viruses are common on pig farms in Guizhou Province, with an incidence rate of 54%, and often as mixed infection. This five-plex PCR method can rapidly detect and differentiate these five viruses.

PRV

PCV2

PRRSV

CSFV

SIV

multiplex PCR

With the large-scale development of China's pig industry, its industrialization and the rapid development of intensive pig herds, mixed viral infections and secondary infections have become increasingly common^[1]. Porcine reproductive and respiratory syndrome virus (PRRSV), Swine influenza virus (SIV), Porcine circovirus type II (PCV2), Classic Swine Fever virus (CSFV) and Porcine Pseudorabies virus (PRV) are five common pathogens of porcine respiratory diseases^[2–3]. These viruses cause porcine respiratory diseases characterized by fever, cough, asthma, difficulty in breathing, increased nasal discharge, loss of appetite, and rapid weight loss. These five viruses also cause varying degrees of damage to the pig's immune system, reduce the pig's resistance to environmental challenges, and cause secondary mixed infections that lead to increased disease complications and pathology. Some can also lead to miscarriage of pregnant sows, stillbirth, birth abnormalities, infertility of young pigs and reproductive disorders^[4–8].

Since Mullis established PCR technology in 1983, the method has been constantly improved and developed. It is widely used in molecular biology and related fields, and has also become the most valuable tool in the detection and diagnosis of animal diseases^[9]. With the continuous development of this technology, the use of multiplex PCR methods for different animal pathogens is becoming increasingly common. Multiplex PCR not only has the advantages of strong specificity, high sensitivity, short detection time, and low cost, but it can also detect multiple pathogens simultaneously, which is useful for the rapid and accurate diagnosis of diseases and the rapid detection of pathogens in a large number of clinical diseases. Zhang et al.^[4] established a double PCR method for the simultaneous detection of Porcine Teschovirus and Porcine Circovirus Type 2. Zeng et al.^[5] established a six-plex PCR method for simultaneously detecting the viral etiological agents of porcine reproductive disorders, PCV2, PRV, Porcine parvovirus (PPV), CSFV, PRRSV, and JEV. In the present study, we undertook to establish a PCR method capable of the simultaneous detection of single or mixed infections of five pathogens, PRRSV, SIV, PCV2, CSFV, and PRV, for the fast and accurate diagnosis of clinical samples.

2.1 Viruses and strains

PRRSV^[6], PCV2, CSFV^[7], and PRV^[8] were identified and preserved in Animal disease Research Laboratory of Guizhou Province. The GenBank accession number for PRV is JX417716. SIV was obtained from a clinically diseased animal from the Guizhou Provincial Institute of Animal Disease Isolation, Identification, and Preservation.

2.2 Reagents and instruments

The TaKaRa MiniBEST Viral RNA/DNA Extraction Kit ver. 4.0 was purchased from Dalian Bao Biological Engineering Co., Ltd. The E.Z.N.A.™ Gel Extraction Kit (50) and Glue Recycling Kit were purchased from Omega Bio-Tek. The PrimeScript One Step RT–PCR Kit ver. 2 and the pMD19-T vector were purchased from Dalian Baosheng Biology Company. The molecular-weight standard DL2000 Marker was purchased from Dalian Bao Biology Company. Isopropyl alcohol, anhydrous ethanol, and other analytical reagents were produced in China. The iCycler Thermal Cycler PCR instrument used was from Bio-Rad.

2.3 Primer and probe design

The multiplex PCR primers were designed with the DNAStar, Oligo, MPprimer, and MFE primer programs at the National Center for Biotechnology Information database for the genes encoding PRV glycoprotein B (gB; AF257079), PCV2 open reading frame 2 (ORF2; NC_005148), PRRSV ORF6 (NC_001961), CSFV C (AY805221), and SIV M (GU086140) The primers were used to amplify the target gene fragments^[9]. The primer sequences are shown in Table 1.

Table 1 Specific primers used to amplify target genes

Virus		Target gene			Primer sequence (5¢–3¢)	Product length (nucleotide)
	PRV			gB	R1： 5¢-ATGGTGGAGGTGCCCG-3¢	192
					F1： 5′-GACCACGCGGTCAATGTCG-3¢
	PCV2		ORF2		R2： 5¢-GGCGGTGGACATGCTGAGAT-3¢	255
					F2： 5¢-TGGGGGATTGTATGGCGGG-3¢
	PRRSV			ORF6	R3： 5¢-GGCCGACTGCTAGGGCTTTT-3¢	364
					F3： 5′-TTCTGCCACCCAACACGAGG-3¢
	CSFV		C		R4： 5¢-GTCAGTAGTTCGACGTGAGCAG-3¢	530
					F4： 5¢-ACCTCGCAGAACTGCACTT-3¢
	SIV			M	R6： 5¢-TTAAAGATGAGCCTTCTGACCGAGG-3¢	981
					F6： 5¢-AGCGCTATGTTGACAAAATGACC-3¢

2.4 Viral nucleic acid extraction

Virally infected cell suspensions (200 μL) and homogenates of the clinical material (200 μL) were processed according to the TaKaRa MiniBEST Viral RNA & DNA Extraction Kit ver. 4.0 instructions for viral RNA and DNA, and stored at −80 °C.

2.5 Single PCR/RT-PCR amplification

Single PCRs or RT–PCRs were established for PRRSV, SIV, PCV2, CSFV, and PRV. The reaction system (25 μL) contained 12.5 μL of 2 × One Step Buffer, 1 μL each of the upstream and downstream primers, 1 μL of the viral template, 1 μL of PrimeScript One Step Enzyme Mix, and 8.5 μL of RNase-free H₂O. The thermal cycling program was reverse transcription at 50 °C for 40 min (PRRSV, SIV, and CSFV only); 95 °C predenaturation for 5 min; 35 cycles of 95 °C for 30 s; 57 °C for 30 s, and 72 °C for 30s; and a final 72 °C extension for 10 min. After completion of the reaction, 5.0 μL of the PCR or RT–PCR product was subjected to electrophoresis on 1% agarose gel.

2.6 Confirmation of five viral products of PCR/RT–PCR

The PCR products of the five viruses were ligated into the pMD19-T vector at 16 °C overnight. TOP10 competent cells were transformed with the ligated products, and the cells were screened with ampicillin (Amp), isopropyl β-d-1-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). White colonies were expanded in culture, and positive bacterial samples were sent to Shanghai Ying Chun Biotechnology Co., Ltd for sequencing.

2.7 Establishment of the best reaction conditions for single PCRs/RT–PCRs

2.7.1 Optimization of single PCRs/RT–PCRs

According to the amplification results of PRRSV, SIV, PCV2, CSFV and PRV genes, the upstream and downstream primers of 0.5, 1, 1.5 or 2 μL of five viruses were detected to determine the optimal reaction system.

The optimum annealing temperature was determined by testing different temperatures for individual PCR/RT-PCR (51°C, 53°C, 55°C, 56°C, 57°C, 58°C, and 60°C).

2.7.2 Testing the sensitivity of the single PCRs/RT–PCRs

The sensitivity of the five virus single PCRs/RT–PCRs was determined with the method of Zhen et al.^[16] and Elnnifro et al.^[10]. The optimal concentrations of PRV, PCV2, PRRSV, CSFV and SIV were determined by protein nucleic acid analyzer and then diluted variously with sterile distilled water (10¹-, 10²-, 10³-, 10⁴-, or 10⁵-fold). An aliquot (1 μL) of each dilution of the viral nucleic acid template was tested with the PCRs/RT–PCRs.

2.8 Establishment of optimal reaction conditions for multiplex PCR (mPCR)

2.8.1 Optimization of the five-plex PCR method

According to the amplification results of PRV, PCV2, PRRSV, CSFV and SIV genes, the different concentrations of 5 pairs of upstream and downstream primers (0.5 μL, 1 μL, 1.5 μL, 2 μL) of the virus were detected to determine the best multiple reaction system.

The optimum annealing temperature was determined by testing various annealing temperatures (51, 51.6, 52.7, 54.5, 56.5, 58.2, 59.3, and 60 °C) in the five-plex PCR.

2.8.2 Testing the specificity of the five-plex PCR method

The specificity of the five-plex PCR was tested with PRV, PCV2, PRRSV, CSFV, SIV nucleic acids.

2.8.3 Five-plex PCR method for the sensitivity test

The sensitivity of the five-plex PCR reaction was determined with the method of Zhen et al. and Elnnifro et al.^[10]. Purified viral nucleic acids of PRV, PCV2, PRRSV, CSFV, and SIV were determined by protein nucleic acid assay and continuously diluted 10-fold with sterile distilled wate (10⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴). PCR amplification was performed for each dilution of the virus template to detect the sensitivity of the assay.

2.8.4 Five-plex PCR analysis of clinical samples

In total, 48 clinical samples were collected from Guizhou Province to test the established five-plex PCR method. Samples of dead pig disease material were repeatedly frozen and thawed for 3 times, thoroughly ground with a tissue homogenizer, centrifuged at 12000 r/min for 5 min, 200 μL was taken from each sample to extract viral DNA or RNA, and analyzed according to the above method.

3.1Single PCR/RT–PCR amplification results

200 μL of PRV, PCV2, PRRSV, CSFV and SIV positive viruses were extracted, and PCR products were extracted according to the instructions of the RNA/DNA extraction kit. PCR/ RT-PCR was used to detect the effectiveness of the designed primers for amplification of the target genes. The results showed that the expression levels of PRV, PCV2, PRRSV, CSFV and SIV positive viruses were 200 μL. The specific bands amplified by single pcr/rt-pcr for PRV, PCV2, PRRSV, CSFV and SIV were consistent with the size of the target fragment. The cloning and sequencing results of each amplified fragment showed that the amplified fragment was the expected gene fragment. The enlarged results are shown in Figure 1.

M: DL2000 Marker; N: negative sample; 1: PCR product of PRV; 2: PCR product of PCV2; 3: PCR product of PRRSV; 4: PCR product of CSFV; 5: PCR product of SIV

Fig. 1 Results of the PCR detection of PRV, PCV2, PRRSV, CSFV, and SIV

3.2Identification of the PCR products

The single PCR/RT–PCR products of PRV, PCV2, PRRSV, CSFV, and SIV were purified, cloned, and sequenced. The results showed that the amplified fragments were the specific gene fragments of each virus.

3.3.1 Optimized conditions for single PCR/RT–PCR

The optimal amounts of primers were determined with a series of experiments in which the concentrations of the primers binding the sample templates were altered. The results showed that when the upstream and downstream primers of PRV and PRRSV were added with 1µL each, and the upstream and downstream primers of PCV2, CSFV and SIV were added with 1.5µL each, the detection effect of PCR/RT-PCR was the most obvious. The optimal reaction system of PRV and PCV2 was 50µL: 10 PCR buffer 5µL, dNTPs1µL, upstream and downstream primers of PRV 1µL, upstream and downstream primers of PCV2 1.5µL, template DNA1µL, rTaq2µL, and ddH2O 39µL and 38µL respectively. The optimal reaction system of PRRSV, CSFV and SIV: reaction system was 25µL: 2×1 Step Buffer 12.5µL; PrimeScript One Step Enzyme Mix 1µL; The upstream and downstream primers of PRRSV were 1µL each, and the upstream and downstream primers of CSFV and SIV were 1.5µL each. Templates 1µL each; RNase Free H2O 9µL, 8µL, 8µL, respectively.

The annealing temperatures of the individual PCRs were tested to determine the optimum reaction conditions. PRRSV showed best amplification at an annealing temperature of 55.0 °C; PRV, PCV2, and CSFV at 56.0 °C; and SIV at 57.0 °C. (1) The optimal conditions for PRV and PCV2 were: 95 °C for 5 min; 35 cycles of 95 °C for 30s, 56.0 °C for 30s, and 72 °C for 30s, s; and 72 °C 10 min. (2) The optimal conditions for PRRSV, CSFV, and SIV were PRV (56 °C)、CSFV (56 °C)、SIV 56 (56 °C) for 30s , 72 °C 30s, 35 cycles; 72 °C for 10 min. The optimization of amplification is shown in Figure 2.

M: DL2000 Marker; 1: 51.0 °C; 2: 53 °C; 3: 55 °C; 4: 56 °C; 5: 57 °C: 6: 58 °C; 7: 60.0 °C

Fig. 2 Optimization of annealing temperaturesfor mPCR of PRV, PCV2, PRRSV, CSFV, and SIV

3.3.2 Testing the sensitivity of single PCRs/RT–PCRs

In the PCRs for single viruses, the minimum amounts of each virus detected were PRV 2.5 pg, PCV2 2.2 pg, PRRSV 3.2 pg, CSFV8.3 pg, and SIV 3.7 pg (Figure 3).

M: DL2000 Marker; 1. diluted 10⁻¹; 2. diluted 10⁻²; 3. diluted 10⁻³; 4. diluted 10⁻⁴; 5. diluted 10⁻⁵

Fig. 3 Sensitivity of the single PCRs/RT–PCRs for PRV, PCV2, PRRSV, CSFV, and SIV

3.4 Establishment of the five-plex PCR method

3.4.1 Optimization of the five-plex PCR method

The optimal primer concentrations were determined by varying the concentrations of the primers used to bind the sample templates. The results showed that the optimum reaction conditions for the five-plex PCR in a 50 μL reaction system were: 0.5 μL (PRV, PCV2), 1.5 μL (PRRSV, CSFV), or 1 μL (SIV) of each forward and reverse primer. The result of the multiplex PCR was the best when the number of reaction cycles was 35 and the annealing temperature was 56.5 °C. The optimal reaction system was a 50 μL reaction volume containing 25 μL of 2 × One Step Buffer, 2 μL of PrimeScript One Step Enzyme Mix, 0.5 μL (PRV, PCV2), 1.5 μL (PRRSV, CSFV), or 1 μL (SIV) of each forward and reverse primer, 1 μL of each template (PRV, PCV2, PRRSV, CSFV, and SIV), and 9 μL of RNase-free H₂O. The optimal cycling parameters were 50 °C for 40 min; 94 °C for 5 min; 35 cycles of 94 °C for 30s, 56.5 °C for 30 s, and 72 °C for 30 s; and 72 °C for 10 min. The optimization of the amplification results is shown in Figure 4.

M: DL2000 Marker; N: negative control; 1: 51.0 °C; 2: 51.6 °C; 3: 52.7 °C; 4: 54.5 °C; 5: 56.5 °C; 6: 58.2 °C; 7: 59.3 °C; 8: 60.0 °C

Fig. 4 Optimization of the annealing temperaturefor mPCR of PRV, PCV2, PRRSV, CSFV, and SIV

3.4.2 Specificity of the five-plex PCR method

No bands were amplified from the PEDV or PPV template with the five-plex PCR, whereas when the PRV, PCV2, PRRSV, CSFV, and SIV templates were amplified, the target fragments were the sizes of the specific bands expected. The results are shown in Figure 5.

M: DL2000 Marker; 1: PCR products of PRV, PCV2, PRRSV, CSFV, and SIV; 2: PCR products of PRV; 3: PCV2; 4: PRRSV; 5: CSFV; 6: SIV; 7: PPV; and 8: PEDV

Fig. 5 Specificity test of the mPCR for PRV, PCV2, PRRSV, CSFV, and SIV

3.4.3 Sensitivity of the five-plex PCR method

In the multiplex PCR, the minimum amounts of the various viruses detected were PRV 18 pg, PCV 22 pg, PRRSV 26 pg, CSFV 48 pg, and SIV 32 pg. The results are shown in Figure 6.

M: DL2000 Marker; 1. diluted 10⁰; 2. diluted 10⁻¹; 3. diluted 10⁻²; 4. diluted 10⁻³; 5. diluted 10⁻⁴; 6. diluted 10⁻⁵

Fig. 6 Sensitivity of the mPCR for PRV, PCV2, PRRSV, CSFV, and SIV

3.4.4 Five-plex PCR method used for clinical samples

The 48 clinical samples tested showed positive results. The PRV-positive rate was 13% (6/48), the PCV2-positive rate was 42% (20/48), the PRRSV-positive rate was 38% (18/48), the CSFV-positive rate was 21% (10/48), and the SIV-positive rate was 40% (19/48). The rate of double infections was 31% (15/48), the rate of triple infections was 17% (8/48), the rate of quadruple infections was 6% (3/48), and the rate of quintuple infections was 0. The correspondence between the test results and the single RT–PCR results was 100%.

In recent years, porcine viral diseases have seriously threatened the world’s large-scale pig industry, and many large-scale pig farms in China have been affected by porcine viral respiratory diseases to varying degrees. PRV, PCV2, PRRSV, CSFV, and SIV are major pathogenic agents of viral respiratory diseases, and the clinical symptoms and pathological changes that occur after infection with these viruses are similar. Therefore, they are difficult to distinguish intuitively. These five viruses are also common in mixed infection, especially of 2–3 kinds of virus. Consequently, the animal diseases caused are complex and the incidence of disease is increasing. These swine diseases entail huge economic losses to the pig industry. A rapid and accurate diagnosis is a prerequisite for the effective prevention and treatment of these diseases^[11]. Meng et al.^[12] have shown that SIV/PRRSV and SIV/PCV-2 double infections and SIV/PRRSV/PRV and SIV/PRRSV/CSFV triple infection are widespread. Bo et al.^[20] showed that there was double infection among PRRSV, CSFV, PCV and PRV in some pig farms in Shanxi Province, and even triple and quadruple mixed infection. The clinical results of this study demonstrate that there are also many mixed viral infections on pig farms in Guizhou Province, so the phenomenon of multiple-virus mixed infections is widespread on pig farms in this country. The rapid and accurate diagnosis of these diseases is essential to ensure a timely and targeted response with drug treatments or immunoprophylactic measures to reduce the risk of disease in pig production, which has important practical significance. Our multiplex PCR method can be used for this purpose, and is suitable for the analysis of large numbers of mixed infections in clinical samples.

Multiplex PCR is based on the development of a single PCR that can simultaneously amplify multiple pathogenic microorganisms or different genes of the same microorganism. Experimental design is more complex and technically challenging than single PCR. To establish a multiplex PCR reaction system, it is necessary to carry out a series of tedious optimization of its main components and reaction conditions, which requires a more comprehensive understanding of PCR technology. After the nucleic acid (DNA and RNA) of the virus can be extracted at the same time, multiple PCR can be used to rapidly detect DNA and RNA viruses, and the deficiency of detection can be gradually made up^[13]. The present study has shown that the optimum annealing temperature for these templates and primers in the mPCR is 56.5 °C, and the sensitivity of detection of the five viruses is lower than the sensitivity of single PCRs/ RT–PCRs.

In 1988, Chamberlain first used the multiplex PCR technology to detect the exons of the human Duchenne muscular dystrophy gene (DMD). Since then, the technique has been extensively studied and applied in many areas, particularly in the field of nucleic acid diagnostics. Pu et al.^[14] developed a quadruple PCR method to simultaneously detect common diseases in pig breeding (porcine respiratory syndrome virus, swine influenza virus, porcine pseudorabies virus and porcine cyclic virus type 2). Xu et al.^[15] successfully established a hexa-plex PCR method to detect PRV, PCV2, PPV, PRRSV, CSFV, and SIV, which include three DNA viruses and three RNA viruses.

In the present study, specific primers were designed to bind the genomes of PRV, PCV2, PRRSV, CSFV, and SIV. The five-plex PCR detection method was established by optimizing the conditions for the amplification of the target genes of PRV, PCV2, CSFV and SIV in order to identify the clinical disease quickly and accurately. This is a practical and rapid detection method with broad application prospects.

Ethical approval and consent to participate: The study does not involve experimental animals, mainly to study the detection method of animal viruses, and the disease materials used are all sick and dead animal tissues

Availability of data and materials: Serial numbers for the study are available in Genebank, and other data and materials are available

Competing interests: I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

funding: Guizhou Province 2017 agricultural research project funding, NO. (2017) 2579

Authors' contributions：Formulation or evolution of overarching research goals and aims, conduct the research process and write the article : Yinming Mao;Development or design of methodology: Deyuan Tang ; Revised article: Zhiyong Zeng ; In charge of experiments: Bin Wang ; Technical Advice: Tao Huang.

Ogawa H, Tairaa O, Hirai T, et al. Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections [J]. J Virological Methods, 2009, 160(1-2):210-214.
Jiagong Zhang, Pathogenesis and prevention and control of porcine respiratory disease syndrome [J]. Animals Breeding and Feed,2008,9:41-42.
Jinwei Chen. Methods of cDNA Chip Assay for Quantitative Detection of Porcine respiratory disease complex related viruses [D]. Heilongjiang Bayi Agricultural University, 2008.
Xiaojie Zhang, Yubing Liu, Deyuan Tang,et al.Establishment and Application of Duplex PCR Assay for the Detection of Porcine Teschovirus and Porcine Circovirus Type 2 [J]. Chinese Journal of Veterinary Drug,2012,46(4):10-13.
Zhiyong Zeng, Zhijie Liu a, Weicheng Wang, et al. Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine[J]. Journal of Virological Methods,2014,208: 102–106.
Liu Xia, Deyuan Tang, Huang Tao, et al.Cloning and constructing of the expression vector of ORF5 gene of PRRSV guizhou isolation strain[J]. Heilongjiang Animal Science and Veterinary Medicine, 2010,(02):102-104.
Huang Tao, Deyuan Tang, Xinqin Ji, et al. Cloning and Construction of the Prokaryotic Expressing Vector and Eukaryotic Expressing Vector of E2 Gene of Classical Swine Fever Virus [J]. Heilongjiang Animal Science and Veterinary Medicine,2009,(10):70-72.
Hao Fei, Deyuan Tang, Chunyan Li, et al.Identification and Pathogenicity Study of Pseudorabies Virus Guizhou-DY Strain[J]. Acta Veterinaria Et Zootechnica Sinica,2013,44(11):1851-1856.
Wang Wen, Wubin Qu, Zhiyong Shen, et al. MPprimer using primers designed and optimized to improve the amplification conditions Multiplex PCR Efficiency Experiment [J]. Progress in Biochemistry and Biophysics,2010,37(3):342-346.
Elnnifro E M, Ashshi A M, Cooper R J,et al. Multiplex PCR: Optimization and application in diagnostic virology[J]. Clincal Microbiology Reviews, 2000, 4(13):559–570.
Hongguang Wang, Shaohong Li, Deyuan Tang, et al. Establishment and application of the triple PCR method for detection of PRV, PCV2 and PPV [J]. Animal Husbandry & Veterinary Medicine,2015,47(1):5-8.
Xueqiong Meng, Liu Qi, Yixiang Chen, et al. Detection of Co-infection with SIV and PRRSV, CSFV, PCV-2 and PRV [J]. Progress in Veterinary Medicine,2006,27(11):84-86.
Guangda Chen. Establishment and Intial Application of Multiplex PCR Assay for Detecting Six Viruses [D]. Northwest A&F University,2012.
Zhao Pu, Yuzhu Zheng, Zhao Kun, et al. Development and Application of a Multiplex Polymerase Chain Reaction for Major Viruses Causing Porcine Respiratory Disease Complex [J]. Acta Agriculturae Boreali-Sinica,2014,29( 3) : 64-67.
Xin-Gang Xu , Guang-Da Chen , Yong Huang, et al. Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses[J]. Journal of Virological Methods,2012,183:69-74.

No competing interests reported.

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Establishment and Application of a Multiplex PCR Method for the Simultaneous Detection of PRV, PCV2, PRRSV, CSFV, and SIV Associated with Porcine Respiratory Disease

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1. Introduction

2. Materials and methods

3 Results

4 Discussion

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