Establishment of a Dual SYBR Green I Fluorescence PCR Assay for

African Swine Fever Virus and Porcine Epidemic Diarrhea Virus

Xinyou Yu

, Tong Li

, Tianzhi Li

, Lin Dong

, Jinliang Wang

4,*

and Zhiqiang Shen

†

Shandong Lvdu Biotechnology Co., Ltd., Binzhou, Shandong 256600, China

College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Postdoctoral Research Station of Shandong Lvdu Biotechnology Co., Ltd., Binzhou, Shandong 256600, China

Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou, Shandong 256600, China

Keywords: African Swine Fever Virus (ASFV), Porcine Epidemic Diarrhea Virus (PEDV), SYBR Green Ⅰ,

Fluorescence PCR.

Abstract: The present study envisaged the development of a fluorescence PCR test for the simultaneous detection of

the African swine fever virus (ASFV) and the porcine epidemic diarrhea virus (PEDV) by designing specific

primers based on the sequences of the p72 gene of ASFV and the N gene of PEDV in GenBank. Subsequently,

the sample loading system and the PCR program was optimized to establish a dual SYBR Green I fluorescence

PCR assay for ASFV and PEDV. Furthermore, the specificity, sensitivity and repeatability of the established

assay were evaluated. Finally, the established PCR assay was tested using clinical samples. The results

demonstrated that the optimal loading amount of each primer in a 20 µL reaction system was: F1 0.5 µL, R1

0.8 µL, F2 1.5 µL and R2 1.2 µL; the optimal PCR program was: reverse transcription at 42 ℃ for 5 min;

pre-denaturation at 95°C for 2 min; and 40 cycles of (denaturation at 94°C for 5 s followed by annealing at

53°C for 25 s, where the fluorescence was collected). The established assay exhibited a good specificity and

did not cross-react with other common swine viruses. The sensitivity of detecting the ASFV and PEDV was

8.8 copies/µL and 3.7 copies/µL, respectively, and the within-run and between-run coefficients of variation

of T

values were not more than 1.0%. The test results of 162 clinical samples using the established PCR

assay were consistent with the reference methods. The dual SYBR Green I fluorescence PCR assay

established in this study for detecting ASFV and PEDV showed high sensitivity and good specificity, and it

can be used for the rapid detection of these two clinical diseases.

1 INTRODUCTION

African swine fever virus (ASFV) can cause African

swine fever (ASF), a severe and highly contagious

infectious disease in swine (Yu and Li, 2018). The

clinical signs include high fever, vomiting,

petechiation of the skin, and bloody diarrhea in pigs

(Jin et al., 2020). Extensive hemorrhage of the various

internal organs was observed in the swine autopsy

(Zhang et al., 2019). ASFV was first detected in a pig

farm in Shenyang, China in August 2018 (Chen et al.,

2018), and it has been prevalent in pig farms in China

for more than 3 years till now. As the clinical variants

emerged, the virulence of the virus diminished, and

the pig farm epidemic eased. The mortality rate of the

infected swine was low in some pig farms (Zhang et

al., 2021). Porcine epidemic diarrhea virus 2 (PEDV)

is a member of the genus Alphacoronavirus in the

family Coronaviridae (Dong et al., 2021). It can

cause porcine epidemic diarrhea (PED) in swine and

is widespread in pig herds worldwide. The clinical

signs include vomiting, watery diarrhea, and

dehydration. The disease has an acute onset, rapid

spread, and a high mortality rate. It can occur

throughout the year, but occurs more often in the

winter and spring (Geng et al., 2021).

Although there are kits for detection of single

pathogen, the operations are cumbersome, and the

cost is high. There is an urgent need to develop a rapid

on-site screening kit for simultaneous detection of

both ASFV and PEDV. Fluorescence polymerase

chain reaction (PCR) has been widely used as a fast,

sensitive and cheap detection method. Compared to

traditional PCR, it has a higher sensitivity and does

not require detection of PCR products by

electrophoresis, reducing the risk of aerosol

Yu, X., Li, T., Li, T., Dong, L., Wang, J. and Shen, Z.

Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus.

DOI: 10.5220/0011594000003430

In Proceedings of the 8th International Conference on Agricultural and Biological Sciences (ABS 2022), pages 5-10

ISBN: 978-989-758-607-1; ISSN: 2795-5893

contamination in the laboratory. To date, there have

been no reports on the dual SYBR Green I

fluorescence PCR method for the simultaneous

detection of ASFV and PEDV. In this pursuit, the

present study aimed to establish a dual SYBR Green

I fluorescence PCR assay to improve the detection

efficiency of ASFV and PEDV.

2 MATERIALS AND METHODS

2.1 Plasmids, Strains, and Field

Samples

The plasmids pMD-p72 and pMD-N containing the

ASFV and PEDV-specific gene fragments,

respectively, were synthesized by General

Biosystems (Anhui) Co., Ltd. The Classical swine

fever virus (CSFV), Porcine reproductive and

respiratory syndrome virus (PRRSV), Porcine

circovirus type 2 (PCV2), Porcine transmissible

gastroenteritis virus (TGEV), Porcine rotavirus (RV),

Porcine pseudorabies virus (PRV) and Porcine

deltacoronavirus (PDCoV) were employed from our

laboratory in the study. A total of 162 pig nasal swab

samples were collected from the pig farms in northern

Shandong from January 2021 to December 2021.

2.2 Primers, Reagents, and

Recombinant Standard Plasmid

Construction

The sequences of the ASFV p72 gene and PEDV

nucleocapsid (N) gene were downloaded from the

GenBank. Specific primers were designed based on

the conserved parts, and further synthesized by

General Biosystems (Anhui) Co., Ltd. The primers

are shown in Table 1. The One-Step TB Green

PrimeScript RT-PCR Kit (Cat. No: RR096A) was

purchased from Takara Bio Inc. (Dalian). The

plasmid miniprep kit was procured from BioTeke

Corporation Co. Ltd. The MyGo Pro quantitative

fluorescence PCR instrument was procured from

Qingdao Buffett Biological Company. The full length

of p72 gene (1941 base pairs) of ASFV (Accession no.

MK554698.1) and N gene (1326 base pairs) of PEDV

(Accession no. MW122505.1) were synthetized and

cloned into the pMD18 vector by Sangon (Shanghai,

China), respectively. The concentration of these

recombinant standard plasmids was determined by

NanoDrop One (ThermoFisher Scientific). The copy

numbers of pMD-p72 and pMD-N plasmids were

2.9×10 9 copies/μL and 1.5×10 9 copies/μL,

respectively. All these plasmids were stored in −20°C

before use.

Table 1: Primers used in the duplex SYBR Green Ⅰ Fluorescence PCR assay.

Virus Gene Name

Primer Sequence (5’→3’)

Position GenBank No.

ASFV p72

F1 CATGGGCAGCTTCAAACGT 391-409

MK554698.1

R1 CAATGGGTCTTCCAAAAG 479-496

PEDV N

F2 TAAGGACCAGCAAATTGGA 135-153

MW122505.1

R2 GTTGTTGCCATTACCACGA 453-471

2.3 Viral Nucleic Acid Extraction

Viral nucleic acid was extracted from each sample

using Simply P Virus RNA/DNA Extraction Kit

(Cat.No:BSC67M1; Hangzhou Bori Technology Co.,

Ltd) according to the manufacturer’s instructions.

The viral DNA/cDNA were stored at − 20 °C for

further study.

2.4 Establishment and Optimization of

Dual Fluorescence PCR Assay

The lyophilized primers F1 and R1 of ASFV, and F2

and R2 of PEDV, respectively, were prepared to

reach a concentration of 15 µmol/L using ultrapure

sterile water. The reaction system was prepared

according to the instructions of the One-Step TB

Green PrimeScript RT-PCR Kit. A total volume of 20

µL was employed for the analysis. This included 10

µL of 2× One-Step TB Green RT-PCR Buffer III, 0.5

µL of PrimeScript RT Enzyme Mix II, and 0.5 µL of

Ex Taq HS, 1 µL of plasmid template pMD-p72, 1 µL

of plasmid template pMD-N, adjusted amounts of

primers for optimization, and adjusted amount of

ultrapure sterile water to make a final volume of 20

µL. The negative control was set up using ultrapure

water to replace the template. After thorough mixing,

the reaction systems were placed in the MyGo Pro

quantitative fluorescence PCR instrument. The PCR

program was set and conducted using various

annealing temperatures such as 51°C, 52°C, 53°C,

54°C, 55°C, 56°C, 57°C, and 58°C.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

2.5 Evaluation of Specificity,

Specificity and Repeatability

To evaluate the specificity of the primer and probe

sets, the synthesized plasmids pMD-p72 and pMD-N

were diluted 10-fold serially, added to the

fluorescence PCR reaction system as templates, and

amplified using the established dual fluorescence

PCR method. For the sensitivity evaluation, the

nucleic acids of CSFV, PPRSV, PPrV, PCV2, TGEV,

PRV, and PDCoV were used as templates to test the

specificity of the established dual fluorescence PCR

assay.

For the evaluation of the repeatability of this

method, the plasmids pMD-p72 and pMD-N samples

of three selected dilution gradients from section 1.6

were used as templates, with three replicates for each

dilution, to perform dual fluorescence PCR. The T

values of the melting curves were analyzed to

calculate the within-run and between-run coefficients

of variation (CV).

2.6 Detection of Clinical Samples

The test samples were collected from various pig

farms in northern Shandong from January 2021 to

December 2021, including healthy hogs and diseased

hogs. In the operation of pig nasal swab collection, a

sterile cotton swab was inserted into the pig’s nasal

cavity and rotated. Then, the swab was withdrawn

from the nostril and placed in a sterilized centrifuge

tube containing 2 mL of normal saline. The tube was

securely closed, put in an ice incubator, and

transported to the laboratory within 24 hours. The

samples were tested using the established ASFV and

PEDV dual SYBR Green I fluorescence PCR. At the

same time, they were tested using the ASFV

fluorescence PCR assay following the national

standard GB/T 18648-2020 and the PEDV

fluorescence PCR assay following the local standard

DB33/T 2254-2020. The test results from our

established PCR were compared to those obtained

from the standard PCR.

3 RESULTS

3.1 Establishment and Optimization of

the Dual Fluorescence PCR Assay

The established optimal loading amounts of primers

were: F1 0.5 µL, R1 0.8 µL, F2 1.5 µL and R2 1.2 µL

in the 20 µL reaction system. The optimal annealing

temperature was 53 °C. The optimal established

fluorescence PCR reaction program was found to be:

reverse transcription at 42 °C for 5 min, pre-

denaturation at 95 °C for 2 min; a total of 40 cycles

of denaturation at 94 °C for 5 s and annealing at 53 °C

for 25 s (where the fluorescence was collected). After

the amplification, the melting curves were drawn and

analyzed, with the increase in temperature to 97 °C at

a rate of 0.1 °C/s and the fluorescence was measured

(Figure 1).

Notes: 1. Plasmid standard template, 2. Negative control.

Figure 1: Melting curves of the dual fluorescent PCR.

Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus

Notes: 1. Plasmid pMD-p72/ORF1, 2. Plasmid pMD-p72, 3. Plasmid pMD-ORF1, 4~10. SFV, PPRSV, PPrV, PCV2, PTGEV,

PRV, and PDCoV.

Figure 2: Amplification specificity of the dual fluorescent PCR assay.

3.2 Evaluation of Sensitivity

The concentration of the plasmid pMD-p72 was

determined to be 8.8×10

copies/µL. The

fluorescence PCR using 10-fold gradient diluted

plasmid showed that the detection limit of plasmid

pMD-p72 was 8.8 copies/µL. The concentration of

plasmid pMD-N was determined to be 3.7×10

copies/µL, and the fluorescence PCR using 10-fold

gradient diluted plasmid showed that the detection

limit of plasmid pMD-N was 3.7 copies/µL.

3.3 Evaluation of Specificity

The established dual SYBR Green I fluorescence

PCR was subjected to the specificity test. The results

showed that the plasmid pMD-p72 had a specific

peak with a T

value of (79.21 ± 0.5) ℃, the plasmid

pMD-N had a specific peak with a T

value of (84.11

± 0.5) ℃, the plasmid pMD-p72/N had double peaks

with T

values of (79.21 ± 0.5) ℃ and (84.11 ±

0.5) ℃. While the other tested pathogens, SFV,

PPRSV, PPrV, PCV2, PTGEV, PRV, and PDCoV, did

not demonstrate specific T

values at (79.21 ± 0.5) ℃

or (84.11 ± 0.5) ℃ (Figure 2). The results indicated

that the established method offered a good specificity

and no cross-amplification reactions with other

pathogenic genes.

3.4 Evaluation of Repeatability

Three different concentrations of 8.8×10

, 8.8×10

and 8.8×10

copies/µL of the plasmid pMD-p72 were

selected from the serial dilution, with three repeats at

each concentration, were subjected to the dual

fluorescence PCR. Similarly, three concentrations of

3.7×10

, 3.7×10

, and 3.7×10

copies/µL of plasmid

pMD-N were selected with three repeats for each

concentration, were subjected to the dual

fluorescence PCR. The T

values of the tested

samples were recorded, and the within-run and

between-run coefficients of variation were calculated,

respectively. The results showed that the within-run

and between-run coefficients of variation of the two

plasmids were not more than 1%, indicating a good

repeatability of the proposed assay (Table 2).

3.5 Diagnostic Performance

The results of the ASFV and PEDV dual SYBR Green

I fluorescence PCR on162 pig nasal swab samples

revealed 0 ASFV positive and 18 PEDV positive,

with no cases of dual infections. Single fluorescence

PCR for the ASFV and PEDV following related

standards, demonstrated consistent results with the

developed dual SYBR Green I fluorescence PCR

assay.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

Table 2: Within-run and between-run repeatability test results of dual fluorescence PCR.

Virus Plasmid

Concentration

N Within-run CV Between-run CV

Mean ± standard deviation CV/% Mean ± standard deviation CV/%

ASFV 8.8×10

8.8×10

79.150±0.254 0.321 78.880±0.165 0.209

79.520±0.148 0.186 79.220±0.425 0.536

78.920±0.632 0.801 79.320±0.612 0.772

PEDV 3.7×10

3.7×10

84.110±0.280 0.333 83.960±0.341 0.406

84.220±0.172 0.204 84.130±0.655 0.779

83.870±0.625 0.745 84.350±0.351 0.416

4 DISCUSSION

China has a huge market for the pork production and

consumption, and the outbreak of ASF and PED have

dealt a severe blow to the swine industry in China.

The accurate and rapid diagnosis of ASF and PED is

an important step in the control and prevention of

these diseases. The fluorescence PCR method has the

advantages of being rapid, high sensitivity and ease

of operation, and it has been widely applied in the

detection of various pathogens. Fluorescence PCR

methods can be divided into two classes: dye-based

and probe-based. Compared with the probe-based

fluorescence PCR, the dye-based assays do not

require the synthesis of expensive probes, thereby

reducing the cost in research and development.

However, the dye-based assays have a higher

requirement for primers, especially for dual PCR

assay, which is the key in the assay establishment.

Another difference from the probe-based method is,

the dye-based fluorescence PCR has an extra step of

melting curve analysis after amplification. With the

increase in the temperature, the melting temperature

at which half of the amplified products dissociate is

the T

peak. Each peak represents a specific product.

By analyzing the difference in T

peaks, multi-

pathogen detection can be realized. Additionally, it

has no special requirements for fluorescent PCR

instruments, and a good versatility. At present, there

is no dual SYBR Green I fluorescence PCR assay

available for detecting ASFV and PEDV.

The ASFV genome consists of a double-

stranded DNA sized 170-190 kb. The p72 gene is

highly conserved, and often serves as a target gene for

the detection of ASFV (Jia et al., 2020). The

nucleocapsid (N) gene encodes the PEDV

nucleocapsid protein, and it is highly conserved and

often used as a target gene for the detection of PEDV

(Pan et al., 2019). The dual SYBR Green I

fluorescence PCR assay proposed in this study for the

simultaneous detection of ASFV and PEDV can

screen the two pathogens rapidly in just 50 minutes.

The key to the successful establishment of the dual

fluorescence PCR assay is the design of primers. In

this study, four primers were found after the screening

of a large number of primers. They exhibited close

annealing temperatures, but did not interfere with

each other. We further optimized the loading amount

of each primer in the reaction system.

The proposed method can reduce costs and

simplify the tedious sample loading steps. The

specific T

peaks of ASFV and PEDV were found to

be at (79.21 ± 0.5) ℃ and (84.11 ± 0.5) ℃,

respectively. Our assay exhibited a good specificity

and did not cross-react with common porcine

pathogens. The limits for detecting ASFV and PEDV

were found to be 8.8 copies/µL and 3.7 copies/µL,

respectively. Tests on different concentrations of

ASFV and PEDV plasmid samples exhibited good

repeatability of the established assay. Neither of the

within-run CV nor between-run CV of T

of the two

plasmids was greater than 1%. Furthermore, among

the clinically obtained 162 pig nasal swabs tested

with the dual fluorescence PCR assay, 0 positive

ASFV cases and 18 positive PEDV cases were found,

and no dual infection cases were found. The 162

samples were subjected to single-plex ASFV and

PEDV PCR assays following related standard and the

results were consistent with the dual SYBR Green I

fluorescence PCR assay. In this study, the T

values

of the two viruses were obtained using the existing

kits and instruments, but the difference between the

two T

values remained unchanged. Although the T

values of the two target products fluctuated, the

amplitudes were not more than 1 °C. In conclusion, a

Establishment of a Dual SYBR Green I Fluorescence PCR Assay for African Swine Fever Virus and Porcine Epidemic Diarrhea Virus

dual SYBR Green Ⅰ fluorescence PCR assay was

established in the present study, which was found to

be time-saving with high sensitivity, high efficiency,

and good repeat-ability. The results indicate that the

proposed assay has an excellent potential to become

a useful laboratory diagnostic tool in the detection of

ASFV and PEDV in clinical samples.

ACKNOWLEDGEMENT

This study was financially supported by Shandong

Major Science and Technology Innovation Project

(No. 2019JZZY020606).

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ABS 2022 - The International Conference on Agricultural and Biological Sciences