Establishment of the Fluorescent RPA Constant Temperature detection method for Novel Muscovy Duck Parvovirus and its primary application

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. Establishment of the Fluorescent RPA Constant Temperature detection method for Novel Muscovy Duck Parvovirus and its primary application [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

Citation:

CHEN W, LIANG Q Z, ZHANG J X, et al. Establishment of the Fluorescent RPA Constant Temperature detection method for Novel Muscovy Duck Parvovirus and its primary application [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

Citation:

CHEN W, LIANG Q Z, ZHANG J X, et al. Establishment of the Fluorescent RPA Constant Temperature detection method for Novel Muscovy Duck Parvovirus and its primary application [J]. Fujian Journal of Agricultural Sciences，2024，39（9）：1−8

PDF( 977 KB)

Establishment of the Fluorescent RPA Constant Temperature detection method for Novel Muscovy Duck Parvovirus and its primary application

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 To establish a fluorescent RPA constant temperature rapid detection method for the novel Muscovy duck parvovirus (N-MDPV), providing a visual rapid detection technology for grassroots. Method This study used the conserved VP3 gene fragment of the novel Muscovy duck parvovirus as the target, and used EXO fluorescent probes to specifically bind to the conserved VP3 gene fragment. Specific RPA amplification primers were designed and the target gene was amplified using recombinant enzyme polymerase amplification technology. A fluorescent type of novel Muscovy duck parvovirus RPA constant temperature rapid detection method was established. Explore and adjust the optimal reaction time and temperature of the reaction system, and test the limit conditions for the specificity and sensitivity of the method. The collected nucleic acid of the disease material was used for detection, and the accuracy of the method was compared with traditional PCR and virus isolation identification methods. Result The optimal reaction temperature for establishing the method in this study is 39 ℃, the optimal reaction time is 30 min, and the lowest sensitivity for nucleic acid detection can reach 10 fg fg·μl⁻¹; The nucleic acid of the novel Muscovy Duck Parvovirus was specifically amplified, but there was no cross reaction between the nucleic acid of duck adenovirus type 3, avian adenovirus type 4, duck circovirus, duck plague virus, duck viral hepatitis virus, duck Tambous virus, and novel duck reovirus, indicating good specificity; The RPA rapid detection method, traditional PCR method, and virus isolation and identification method established in this study were used to detect the nucleic acid samples of 38 collected duck tissue samples. The results showed that the positive rates were 36.8% (14/38), 36.8% (14/38), and 31.6% (12/38), respectively; The samples that tested positive for RPA showed positive results through PCR and virus isolation and identification methods, with a positive coincidence rate of 100%. The positive coincidence rate between RPA detection method and traditional PCR method in clinical sample testing is 100%. Conclusion This method can be well applied to the large-scale clinical sample detection of the novel muscovy duck parvovirus in grassroots areas lacking corresponding detection equipment, providing a technical means for the visual and rapid detection of the novel muscovy duck parvovirus.
- Novel Muscovy duck parvovirus,
- RPA Constant temperature detection,
- recombinase polymerase amplification technology

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)