Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (1): 197-207.doi: 10.3864/j.issn.0578-1752.2022.01.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Development and Application of a Real-Time Fluorescent RPA Diagnostic Assay for African Swine Fever

ZHANG JingYuan1,2(),MIAO FaMing2,CHEN Teng2,LI Min1(),HU RongLiang2()   

  1. 1School of Life Sciences, Ningxia University, Yinchuan 750021
    2Veterinary Research Institute, Institute of Military Medical Sciences, Academy of Military Sciences, Changchun 130122
  • Received:2020-11-16 Accepted:2021-02-03 Online:2022-01-01 Published:2022-01-07
  • Contact: Min LI,RongLiang HU E-mail:zjyuanff27@163.com;lim@nxu.edu.cn;ronglianghu@hotmail.com

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Abstract:

【Objective】 After the first outbreak of African Swine Fever (ASF) in Shenyang, China in 2018, it has rapidly spread to the whole country, severely hitting the pig industry. This study aimed to establish an optimized nucleic acid testing technique for African Swine Fever Virus (ASFV), so as to provide a fast and accurate method for early diagnosis and accurate treatment of ASF outbreaks. 【Method】 Appropriate primers and probes were designed and screened for the conserved gene B646L (p72) of ASFV, and a real-time fluorescent RPA assay based on recombinase polymerase amplification (RPA) was established. The reaction system, reaction conditions and sample treatment steps were optimized. Specificity and sensitivity of the optimized detection method were evaluated by using quality controls. In addition, 1 009 clinical samples were tested by the optimized real-time RPA, after which the results were further confirmed by the real time PCR recommended by OIE and through virus isolation. 【Result】 A pair of primers-probe combinations was successfully screened, and a real-time fluorescence RPA for detection of ASFV p72 gene was developed. The total volume of optimized reaction system was 25 μL. The reaction conditions were set as 39℃ 10 s, 39℃ 20 s, 40 cycles on the fluorescence quantitative PCR instrument, and the whole amplification reaction needs about 20 min. The analysis method at room temperature could replace the traditional nucleic acid extraction method, thus the whole process of sample treatment, nucleic acid amplification and result reading could be completed in 30 min. Specific evaluation showed that the real-time RPA was negative for porcine parvovirus (PPV), pseudorabies virus (PRV), circovirus type1/2 (PCV1/2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV); the sensitivity evaluation showed that the assay could detect type I/II/IX ASFV samples, and could detect 10 copies/μL of ASFV positive simulated blood samples and 1﹕103.0 dilution of positive clinical samples, which was as sensitive as the OIE-recommended qPCR method. Seventeen out of 1 009 clinical samples were tested positive using the real-time RPA, with the same results as by qPCR, 17 positive cultures were obtained from virus isolation. 【Conclusion】 A real-time RPA diagnostic method for ASF was developed, which was proved to be simple, less time consuming with high sensitivity and specificity, providing a new, simple, specific and rapid diagnostic method for ASF.

Key words: African swine fever virus, recombinase polymerase amplification, real-time fluorescent RPA, nucleic acid detection, diagnosis

Table 1

Amplification system for real-time RPA"

序号
Number		 组分
Components		 体积
Volume (μL)
1 Primer F 2.1
2 Primer R 2.1
3 Exo Probe 0.6
4 Rehydration buffer 29.5
5 Water 11.2
6 Template 1 1
7 MgOAc（280 mmol·L-1） 1.25 1.25
合计Total 25 25

Table 2

Optimization of Real-time RPA amplification system"

体系A System A 体系B System B
组分 Components 体积 Volume(μL) 组分 Components 体积 Volume(μL)
Primer F 2.1 Rehydration Mix 40（2 reactions）
Primer R 2.1
Exo Probe 0.6
Rehydration buffer 29.5
Water 11.2
将溶解后的反应体系等分至两个反应管 Equal distribution of the dissolved amplification system into two tubes
Template 1 1 Template 1 1
MgOAc（280 mmol·L-1） 1.25 1.25 MgOAc-B 4 4
合计 Total 25 25 合计 Total 25 25

Fig. 1

Screening of primers-probe combination for real-time RPA Pa: Set a; Pb: Set b; Pc: Set c; 1: Strong positive control; 2: Weak positive control; 3: Negative control"

Table 3

Primers and probe of Real-time fluorescent RPA"

名称 Name 序列（5′-3′）Sequence (5′-3′)
EXO-F TAATAGCAGATGCCGATACCACAA
EXO-R TTACATACCCTTCCACTACGGAGGC
EXO-P GTCCCAACTAATATAAAATTCTCTTGCTC/i6FAMdT/G/idSp/A/iBHQ1dT/ACGTTAATATGACCAC-P

Fig. 2

Optimization of reaction temperature and time for real-time RPA 1: Strong positive control; 2: Weak positive control; 3: Negative control"

Fig. 3

Optimization of the amplification system"

Table 4

Comparison of different sample treatment methods"

处理方法
Treatment		 所需温度
Temperature		 操作步骤
Operation Steps		 处理时间
Time costs		 Ct值Ct Value (2Ct≈1min)
N WP SP
无处理Untreatment / / 0 min / 33.92 19.39
磁珠法
Magnetic beads extraction		 RT 结合,清洗,洗脱
Combination, Wash, Elution		 10 min / 22.23 17.76
柱提取法
Column extraction		 RT 裂解,沉淀,离心,洗脱
Lysis, Precipitation, Centrifugation, Elution		 30 min / 23.54 14.02
裂解法
Lysis		 RT 裂解,离心
Lysis, Centrifugation		 5 min / 24.07 14.25
煮沸法
Boiling		 100℃,4℃ 煮沸,冷藏,离心
Boiling, Refrigeration, Centrifugation		 10 min / 22.05 16.96

Fig. 4

Results of specific samples tested by real-time RPA and qPCR"

Fig. 5

Results of sensitivity samples detected by real-time RPA and qPCR"

Table 5

Results of clinical samples with different dilutions tested by real-time RPA and qPCR"

样品种类
Samples		 稀释倍数
Dilutions		 检测结果Results
实时RPA Real-time RPA 荧光定量PCR rt-PCR
Ct值Ct value 判定Read Ct值Ct value 判定Read
脾脏
Spleen		 1:101.0 30.49 + 28.00 +
1:102.0 35.55 + 30.23 +
1:103.0 38.35 + 34.71 +
1:104.0 / - / -
淋巴结
Lymph node		 1:101.0 28.58 + 29.44 +
1:102.0 34.61 + 30.11 +
1:103.0 39.33 + 37.21 +
1:104.0 / - / -
血清
Serum		 1:101.0 24.76 + 27.32 +
1:102.0 29.68 + 32.33 +
1:103.0 32.12 + 35.56 +
1:104.0 / - / -

Fig. 6

Virus isolation and immunofluorescent staining of positive samples"

Table 6

Summary of positive clinical sample test results"

编号
Number		 样品种类
Sample type		 检测结果Results
实时RPA Real-time PRA qPCR 病毒分离Virus isolation
Ct值Ct Value 判定Read Ct值Ct Value 判定Read 判定Read
34 脾脏Spleen 20.60 + 19.37 + +
35 脾脏Spleen 12.50 + 22.60 + +
36 脾脏Spleen 20.74 + 18.71 + +
37 脾脏Spleen 32.82 + 29.28 + +
38 脾脏Spleen 14.72 + 20.17 + +
117 淋巴结Lymph node 12.83 + 19.77 + +
246 脾脏Spleen 17.48 + 27.07 + +
356 淋巴结Lymph node 19.92 + 18.72 + +
766 脾脏Spleen 25.27 + 19.17 + +
767 脾脏Spleen 26.85 + 23.47 + +
770 血清Serum 14.85 + 20.01 + +
771 血清Serum 17.23 + 26.61 + +
841 淋巴结Lymph node 16.40 + 26.57 + +
900 血清Serum 14.55 + 21.09 + +
913 血清Serum 35.46 + 38.45 + +
943 脾脏Spleen 15.31 + 22.72 + +
968 淋巴结Lymph node 20.16 + 26.22 + +

Table 7

Comparison of operation process between real-time RPA and qPCR"

检测方法
Methods		 扩增温度
Temperature		 扩增设备
Instrument		 扩增时间
Time (min)
Real-time RPA 37℃ 等温扩增仪或实时荧光PCR仪
Isothermal amplification instrument real time
fluorescence PCR instrument		 20
qPCR 95℃,
60℃		 实时荧光PCR仪
Real time fluorescence PCR instrument		 >60
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