Establishment and preliminary application of fluorescent RT-RAA diagnostic method for porcine epidemic diarrhea virus
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摘要:
目的 建立一种快捷、灵敏、简便检测猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）的RT-RAA检测方法，以提高猪流行性腹泻病毒临床检测效率。 方法 针对PEDV S基因片段保守区设计引物和探针，构建标准质粒PEDV-S，通过特异性、敏感性、重复性测定及条件优化，建立检测PEDV重组酶介导链置换核酸扩增荧光法（RT-RAA）。 结果 在42 ℃恒温作用20 min的条件下，建立的检测方法对检测猪传染性胃肠炎病毒（porcine transmissible gastroenteritis virus, TGEV）、猪瘟病毒（classical swine fever virus, CSFV）、伪狂犬病病毒（porcine pseudorabies virus, PRV）、猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus, PRRSV）和猪轮状病毒（porcine rotavirus, PoRV）等猪源病毒均为阴性，猪流行性腹泻病毒（PEDV）为阳性；最低检出限为4.43×102拷贝·μL−1的标准质粒；重复性结果显示，相同浓度标准质粒的差异性很小；利用建立的RT-RAA方法检测40份猪病毒样本，阳性率均为7.5%（3/40），与实时荧光定量PCR检测结果相同。 结论 本研究建立的PEDV RT-RAA检测方法具有快速简便、耗时短、特异性高、灵敏度强和重复性好等特点，适用于对PEDV的快速诊断。 Abstract:Objective To establish a quick, simple, sensitive detection of porcine epidemic diarrhea virus (PEDV) the RT- RAA detection method, to improve the efficiency of porcine epidemic diarrhea virus clinical detection. Method Primers and probes were designed for the conserved region of PEDVS gene fragment, and a standard plasmid PEDV-S was constructed. Through specificity, sensitivity, repeatability and condition optimization, a recombinant enzyme-mediated chain replacement nucleic acid amplification fluorescence assay (RT-RAA) for detection of PEDV was established. Result Under the condition of constant temperature at 42 ℃ for 20 min,The established assay is effective for the detection of porcine transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV) and other porcine viruses were negative, porcine epidemic diarrhea virus (PEDV) was positive.The minimum detection limit was 4.43× 102 copies·μL−1 standard plasmid.The reproducibility results showed that there was little difference between the standard plasmids with the same concentration.The positive rate of 40 swine virus samples was 7.5% (3/40) by RT-RAA method, which was the same as that of real-time fluorescence quantitative PCR. Conclusion The RT-RAA detection method for PEDV established in this study is suitable for the rapid diagnosis of PEDV due to its rapid and simple detection, short time-consuming, high specificity, strong sensitivity and good reproducibility. -
Key words:
- porcine epidemic diarrhea virus /
- S protein /
- RT-RAA
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图 1 引物及探针所在S基因保守区域
Figure 1. The conserved region of the S gene where the primers and probes are located
图 2 PEDV S 基因PCR扩增
M：DL2000 Marker；1～4：PEDV S 基因扩增；5：阴性对照。
Figure 2. PCR amplification of the PEDV S gene
M：DL2000 Marker；1–4: PEDV S gene amplification; 5: negative control.
图 3 荧光型RT-RAA引物筛选
1：F3/R3；2：F3/R2；3：F1/R1；4：F2/R3；5：F3/R1；6：F1/R2；7：F2R2；8：F1/R3；9：F2/R1；10：阴性对照。
Figure 3. Fluorescent special-pull primer screening
1: F3/R3; 2: F3/R2; 3: F1/R1; 4: F2/R3; 5: F3/R1; 6: F1/R2; 7: F2/R2; 8: F1/R3; 9: F2/R1; 10: negative control.
图 4 荧光型RT-RAA反应温度的筛选
1～3分别为42、39和37 ℃。
Figure 4. Screening of fluorescent RT-RAA reaction temperatures
1–3was 42, 39 and 37 °C, respectively.
图 5 荧光型RT-RAA反应时间的筛选
1～3分别为反应20、25、17min。
Figure 5. Screening of fluorescent RT-RAA reaction time
1–3 was 20, 25 and 17 min for reaction.
图 6 荧光型RT-RAA特异性试验
1：PEDV-S；2～8：TGEV、CSFV、PRV、PCV、PRRSV、PoRV、阴性对照。
Figure 6. Fluorescent RT-RAA specificity test
1: PEDV-S; 2–8: TGEV, CSFV, PRV, PCV, PRRSV, PoRV, negative control.
图 7 荧光型RT-RAA的重复性检测
1～3：1×105拷贝·μL−1；4～6：1×103拷贝·μL−1；7～9：1×102拷贝·μL−1。
Figure 7. Repeatability detection of fluorescent RT-RAA
1–3: 1×105 copies·μL−1; 4–6: 1×103 copies·μL−1; 7–9: 1×102 copies·μL−1.
图 8 荧光型RT-RAA敏感性试验
1～7：标准质粒依次为106～100 拷贝·μL−1；8：阴性对照。
Figure 8. Fluorescence RT-RAA sensitivity test
1–7: the standard plasmid was 106–100 copies·μL−1; 8: negative control.
表 1 疑似猪流行性腹泻病料收集的来源信息
Table 1. Source information of sample collection for suspected porcine epidemic diarrhea
地区
Region样本数（份）/来源猪场（个）
Samples/Farms宁德 Ningde 5/2 三明 Sanming 6/2 龙岩 Longyan 10/6 漳州 Zhangzhou 8/3 莆田 Putian 11/6 合计 total 40/19 
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表 2 引物和探针
Table 2. Primers and probes
名称
Name序列（5′-3′）
SequencePEDV S-DF AAATCTGGCAGTATTGGCTAC PEDV S-DR ATCGGCTGAAAGAATGTCC PEDV S-F1 TATTCCCACCAACTTTAGTATGAGTATTAG PEDV S-F2 GTATTCCCACCAACTTTAGTATGAGTATTA PEDV S-F3 AGTATTCCCACCAACTTTAGTATGAGTATT PEDV S-R1 TAATGCTGACTCTATGGTCTTACATGCTGC PEDV S-R2 GTTGTAATGCTGACTCTATGGTCTTACATG PEDV S-R3 TGTAATGCTGACTCTATGGTCTTACATGCT PEDV S-P GACAGAATATTTACAGCTTTACAACACGCC(i6FAMdT)(THF)(iBHQ1dT)TAGTGTTGATTGTGC-C3spacer
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表 3 临床样本的检测
Table 3. Detection of clinical samples
方法
Method阳性样品
Number of
positives/份阴性样品
Number of
negatives/份总数
Total/份阳性率
Positivity
rate/%RT-RAA 3 37 40 7.5 RT-qPCR 3 37 40 7.5
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[1] JUNG K, SAIF L J, WANG Q H. Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control [J]. Virus Research, 2020, 286: 198045. doi: 10.1016/j.virusres.2020.198045 [2] LEE C. Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus [J]. Virology Journal, 2015, 12: 193. [3] ZHOU Z J, QIU Y, GE X Y. The taxonomy, host range and pathogenicity of coronaviruses and other viruses in the Nidovirales order [J]. Animal Diseases, 2021, 1(1): 5. doi: 10.1186/s44149-021-00005-9 [4] ZHANG Y Z, CHEN Y W, ZHOU J, et al. Porcine epidemic diarrhea virus: An updated overview of virus epidemiology, virulence variation patterns and virus-host interactions [J]. Viruses, 2022, 14(11): 2434. doi: 10.3390/v14112434 [5] WU X H, LIU Y J, GAO L G, et al. Development and application of a reverse-transcription recombinase-aided amplification assay for porcine epidemic diarrhea virus [J]. Viruses, 2022, 14(3): 591. [6] PEWLAOO S, PHANTHONG S, KONG-NGOEN T, et al. Development of a rapid reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow method for detecting porcine epidemic diarrhoea virus [J]. Biology, 2022, 11(7): 1018. doi: 10.3390/biology11071018 [7] LI G, WU M L, LI J H, et al. Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method [J]. Journal of Virological Methods, 2021, 292: 114096. doi: 10.1016/j.jviromet.2021.114096 [8] MAO L J, YING J X, SELEKON B, et al. Development and characterization of recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of monkeypox virus [J]. Viruses, 2022, 14(10): 2112. doi: 10.3390/v14102112 [9] NIE M C, ZHOU Y C, LI F Q, et al. Epidemiological investigation of swine Japanese encephalitis virus based on RT-RAA detection method [J]. Scientific Reports, 2022, 12(1): 9392. doi: 10.1038/s41598-022-13604-4 [10] ZHAO S, ZHANG Q Q, WANG X Y, et al. Development and performance of recombinase-aided amplification (RAA) assay for detecting Schistosoma haematobium DNA in urine samples [J]. Heliyon, 2023, 9(12): e23031. doi: 10.1016/j.heliyon.2023.e23031 [11] REN J, ZU C C, LI Y, et al. Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses [J]. Frontiers in Microbiology, 2024, 15: 1380849. [12] NIU J W, LI J H, GUAN J L, et al. Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses [J]. Frontiers in Veterinary Science, 2022, 9: 1033864. doi: 10.3389/fvets.2022.1033864 [13] LIANG W, ZHOU D N, GENG C, et al. Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia [J]. PeerJ, 2020, 8: e10114. doi: 10.7717/peerj.10114 [14] ZHANG H L, HAN F F, YAN X G, et al. Prevalence and phylogenetic analysis of spike gene of porcine epidemic diarrhea virus in Henan province, China in 2015-2019 [J]. Infection, Genetics and Evolution, 2021, 88: 104709. doi: 10.1016/j.meegid.2021.104709 [15] LI Z W, MA Z Q, LI Y, et al. Porcine epidemic diarrhea virus: Molecular mechanisms of attenuation and vaccines [J]. Microbial Pathogenesis, 2020, 149: 104553. doi: 10.1016/j.micpath.2020.104553 [16] 俞正玉, 逄凤娇, 孙冰, 等. 猪流行性腹泻病毒变异株RT-PCR检测方法的建立 [J]. 江苏农业科学, 2018, 46(1):116−118.YU Z Y, PANG F J, SUN B, et al. Establishment of RT-PCR method for detection of porcine epidemic diarrhea virus mutant [J]. Jiangsu Agricultural Sciences, 2018, 46(1): 116−118. (in Chinese) [17] SONG W B, FENG Y X, ZHANG J L, et al. Development of a multiplex reverse transcription-quantitative PCR (qPCR) method for detecting common causative agents of swine viral diarrhea in China [J]. Porcine Health Management, 2024, 10(1): 12. doi: 10.1186/s40813-024-00364-y [18] LI C H, LIANG J L, YANG D, et al. Visual and rapid detection of porcine epidemic diarrhea virus (PEDV) using reverse transcription loop-mediated isothermal amplification method [J]. Animals, 2022, 12(19): 2712. doi: 10.3390/ani12192712 [19] WANG Y S, NIE M C, DENG H D, et al. Establishment of a reverse transcription recombinase-aided amplification detection method for porcine group a rotavirus [J]. Frontiers in Veterinary Science, 2022, 9: 954657. doi: 10.3389/fvets.2022.954657 [20] XIA W L, CHEN Y, DING X, et al. Rapid and visual detection of type 2 porcine reproductive and respiratory syndrome virus by real-time fluorescence-based reverse transcription recombinase-aided amplification [J]. Viruses, 2022, 14(11): 2526. doi: 10.3390/v14112526 [21] WANG S C, ZHUANG Q Y, JIANG N, et al. Reverse transcription recombinase-aided amplification assay for avian influenza virus [J]. Virus Genes, 2023, 59(3): 410−416. -
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