A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus

CHEN Wei; LIANG Qizhang; ZHANG Jiaxue; JIAO Wenlong; LIN Yongqiang; LIU Rongchang; FU Qiuling; FU Guanghua; ZHU Ting; HUANG Yu

Article Contents

Article Navigation > Fujian Journal of Agricultural Sciences > 2024 >

CHEN W, LIANG Q Z, ZHANG J X, et al. A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

Citation:

CHEN W, LIANG Q Z, ZHANG J X, et al. A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

CHEN W, LIANG Q Z, ZHANG J X, et al. A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

Citation:

CHEN W, LIANG Q Z, ZHANG J X, et al. A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

PDF( 981 KB)

A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus

CHEN Wei^{1, 2
,},
LIANG Qizhang^{2, 3
,},
ZHANG Jiaxue^{1, 2},
JIAO Wenlong^{1, 2},
LIN Yongqiang^{1, 2},
LIU Rongchang^{2, 3},
FU Qiuling^{2, 3},
FU Guanghua^{2, 3},
ZHU Ting^{1
,
,},
HUANG Yu^{2, 3
,
,}

1.
College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry university, Fuzhou, Fujian　350002, China
2.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural sciences, Fuzhou, Fujian　350013, China
3.
Fujian Key Laboratory for Avian Diseases Control and Prevention, Fuzhou, Fujian　350013, China

Received Date: 2024-05-09
Rev Recd Date: 2024-08-01

Available Online: 2024-10-22

Abstract

Abstract

Objective A constant temperature fluorescent RPA assay for detecting novel Muscovy duck parvovirus (N-MDPV) was developed. Method EXO fluorescent probes were specifically bound to the targeted conserved VP3 fragment of N-MDPV. RPA amplification primers were designed to amplify the segment by using recombinant enzyme polymerase. An assay for detecting N-MDPV was established with reaction time and temperature optimized and specificity, sensitivity, and accuracy tested on the collected nucleic acid of disease material in comparison with traditional PCR and virus isolation identification methods. Result The optimized assay reaction temperature and time were 39 ℃ for 30m to achieve a lowest sensitivity for nucleic acid detection at 10 fg·μl⁻¹. The nucleic acid of N-MDPV was specifically amplified without any cross reaction with those of duck adenovirus type 3, avian adenovirus type 4, duck circovirus, duck plague virus, duck viral hepatitis virus, duck Tambousu virus, and novel duck reovirus. Along with the conventional PCR and the virus isolation and identification methods, the newly developed assay detected the nucleic acid on 38 duck tissue specimens with a positive rate of 36.8% (14/38) in comparison to those at 36.8% (14/38) for the PCR and 31.6% (12/38) for the isolation and identification method. In addition, 100% coincidence rates of the assay and the two other methods on positive samples as well as of the assay and the PCR on the positive clinical samples were secured. Conclusion The new RPA method to rapidly and visually detect N-MDPV demonstrated to be highly specific, sensitive, and accurate. It was deemed appropriate for clinical applications.

FullText(HTML)

References(12)

References

[1]	林世棠, 郁晓岚, 陈炳钿, 等. 一种新的雏番鸭病毒性传染病的诊断 [J]. 中国畜禽传染病, 1991, 13(2):25−26. LIN S T, YU X L, CHEN B D, et al. Diagnosis of a new viral infectious disease in Muscovy duck [J]. Chinese Journal of Preventive Veterinary Medicine, 1991, 13(2): 25−26. (in Chinese)
[2]	胡奇林, 吴振兖, 周文谟, 等. 雏番鸭细小病毒病的流行病学调查 [J]. 中国兽医杂志, 1993, 29(6):7−8. HU Q L, WU Z Y, ZHOU W M, et al. Epidemiological investigation of parvovirus disease in Muscovy ducks [J]. Chinese Journal of Veterinary Medicine, 1993, 29(6): 7−8. (in Chinese)
[3]	黄瑜, 万春和, 傅秋玲, 等. 新型番鸭细小病毒的发现及其感染的临床表现 [J]. 福建农业学报, 2015, 30(5):442−445. doi: 10.3969/j.issn.1008-0384.2015.05.004 HUANG Y, WAN C H, FU Q L, et al. The identity and clinic infectious symptoms of the new genotype Muscovy duck parvovirus [J]. Fujian Journal of Agricultural Sciences, 2015, 30(5): 442−445. (in Chinese) doi: 10.3969/j.issn.1008-0384.2015.05.004
[4]	孟婷, 朱善元, 夏文龙, 等. 番鸭细小病毒LAMP检测方法的建立 [J]. 中国动物传染病学报, 2019, 27(1):25−29. MENG T, ZHU S Y, XIA W L, et al. Development of a loop-mediated isothermal amplification assay for detection of Muscovy duck parvovirus [J]. Chinese Journal of Animal Infectious Diseases, 2019, 27(1): 25−29. (in Chinese)
[5]	EULER M, WANG Y J, OTTO P, et al. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis [J]. Journal of Clinical Microbiology, 2012, 50(7): 2234−2238. doi: 10.1128/JCM.06504-11
[6]	刘孟啸, 蔡姝, 刘建钗, 等. 鸭腺病毒3型荧光RPA恒温快速检测方法的建立与应用 [J]. 中国兽药杂志, 2023, 57(11):8−15. LIU M X, CAI S, LIU J C, et al. Rapid detection of duck adenovirus type 3 based on isothermal recombinase polymerase amplification assay [J]. Chinese Journal of Veterinary Drug, 2023, 57(11): 8−15. (in Chinese)
[7]	袁嘉康, 李林岳, 庞俊增, 等. 猪流行性腹泻病毒RPA-LFD检测方法的建立 [J]. 中国兽医学报, 2023, 43(6):1127−1132. YUAN J K, LI L Y, PANG J Z, et al. Establishment of RPA-LFD method for detection of porcine epidemic diarrhea virus [J]. Chinese Journal of Veterinary Science, 2023, 43(6): 1127−1132. (in Chinese)
[8]	ABD EL WAHED A, EL-DEEB A, EL-THOLOTH M, et al. A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus [J]. PLoS One, 2013, 8(8): e71642. doi: 10.1371/journal.pone.0071642
[9]	王建昌, 王金凤, 刘立兵, 等. 非洲猪瘟病毒RPA等温检测方法的建立 [J]. 中国动物检疫, 2016, 33(7):78−81,94. doi: 10.3969/j.issn.1005-944X.2016.07.024 WANG J C, WANG J F, LIU L B, et al. Rapid and sensitive detection of African swine fever virus by recombinase polymerase amplification [J]. China Animal Health Inspection, 2016, 33(7): 78−81,94. (in Chinese) doi: 10.3969/j.issn.1005-944X.2016.07.024
[10]	DAHER R K, STEWART G, BOISSINOT M, et al. Recombinase polymerase amplification for diagnostic applications [J]. Clinical Chemistry, 2016, 62(7): 947−958. doi: 10.1373/clinchem.2015.245829
[11]	万春和, 傅秋玲, 陈翠腾, 等. 基因重组型番鸭细小病毒FJM3的全基因组特征 [J]. 中国兽医学报, 2016, 36(11):1836−1841. WAN C H, FU Q L, CHEN C T, et al. Molecular characterization of the genome for recombinant Muscovy duck parvovirus strain FJM3 [J]. Chinese Journal of Veterinary Science, 2016, 36(11): 1836−1841. (in Chinese)
[12]	刘家森, 姜骞, 司昌德, 等. 番鸭细小病毒与鹅细小病毒PCR鉴别诊断方法的建立 [J]. 中国兽医科学, 2007, 37(6):469−472. LIU J S, JIANG Q, SI C D, et al. Establishment of PCR assay for differentiation of Muscovy duck parvovirus from goose parvovirus [J]. Veterinary Science in China, 2007, 37(6): 469−472. (in Chinese)