[1]陈翠翠,梁焕坤,赖宏锐,等.犬细小病毒时间分辨免疫荧光分析方法的建立及初步应用[J].中国预防兽医学报,2021,(02):165-170.[doi::10.3969/j.issn.1008-0589.202005022]
 CHEN Cui-cui,LIANG Huan-kun,LAI Hong-rui,et al.Establishment and preliminary application of time-resolvedimmunofluorescence analysis method for Canine parvovirus[J].Chinese journal of preventive veterinary medicine,2021,(02):165-170.[doi::10.3969/j.issn.1008-0589.202005022]
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犬细小病毒时间分辨免疫荧光分析方法的建立及初步应用
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2021年02
页码:
165-170
栏目:
诊断和检测技术
出版日期:
2021-03-16

文章信息/Info

Title:
Establishment and preliminary application of time-resolved
immunofluorescence analysis method for Canine parvovirus
文章编号:
:1008-0589(2021)02-0165-06
作者:
 陈翠翠梁焕坤赖宏锐钟树海黎杰星李来庆*
 (广州优迪生物科技股份有限公司,广东广州510663)
Author(s):
 CHEN Cui-cui LIANG Huan-kun LAI Hong-rui ZHONG Shu-hai LI Jie-xing LI Lai-qing*
 (Guangzhou Youdi Biotechnology Co., Ltd., Guangzhou 510663, China)
关键词:
:犬细小病毒双抗体夹心时间分辨免疫荧光分析
Keywords:
: Canine parvovirus double-antibody sandwich time-resolved immunofluorescence analysis (TRFIA)
分类号:
S852.65
DOI:
:10.3969/j.issn.1008-0589.202005022
文献标志码:
A
摘要:
:为建立犬细小病毒(CPV)的时间分辨免疫荧光分析(TRFIA)方法并制备试剂盒,本实验采用抗CPV
单克隆抗体(MAb)7D5作为包被抗体,Eu3+标记的MAb 2F7作为检测抗体,对各反应条件优化后制备双抗体夹心
TRFIA试剂盒。并评价了其灵敏度、准确度、特异性、重复性和稳定性。利用该试剂盒与RT-PCR方法同时检测
了疑似患病犬的临床粪便、呕吐物、血液样品(各60份)和CPV阴性犬这3种临床样品各10份,比较并计算二者的
符合率。优化结果显示, 最佳的TRFIA 反应条件为: MAb 7D5 的包被浓度为15 μg/mL; Eu3 + 标记试剂与标
记MAb 2F7(2.5 mg/mL)最佳体积分别为70 μL和30 μL;反应体系为25 μL CPV标准品或待测样品+200 μL分析缓冲
液+200 μL Eu3+-检测抗体+200 μL增强液。灵敏度试验显示TRFIA方法对CPV灭活病毒检测灵敏度为0.83 ng/mL。
准确度试验结果显示,不同浓度CPV标准品稀释(1:2、1:4、1:8)后的稀释回收率在89.25%~100.8%;特异性试
验结果显示,该方法除对不同亚型CPV的检测结果均为阳性外,对犬瘟热病毒、犬副流感病毒、犬腺病毒I型、
犬冠状病毒、猫细小病毒、水貂肠炎病毒等检测均为阴性,无明显交叉反应;对高、中、低3种不同浓度CPV的重
复性试验结果显示,批内变异系数为2.34%~4.76%,批间变异系数为2.38%~5.10%;稳定性试验结果显示,该试剂
盒至少可在4 ℃稳定保存6个月,37 ℃保存7 d;临床样品的检测结果显示,利用该方法从大部分疑似患病犬的粪
便、血液以及呕吐物中均检测到了CPV,而CPV阴性犬这3类样品的检测结果均为阴性。本研究建立的TRFIA法和
RT-PCR法检测结果一致,符合率达到100%。上述结果表明,本研究制备的CPV TRFIA试剂盒灵敏度准确度高、特
异性强、重复性、稳定性好,且可检测的临床样品种类多,为临床样品的检测提供有效技术手段。
Abstract:
To establish a time-resolved immunofluorescence analysis (TRFIA) method for Canine parvovirus (CPV) and
prepare the corresponding kits anti-CPV monoclonal antibody (MAb) 7D5 as the coating antibody and Eu3 + labeled MAb 2F7 as
the detection antibody were used to prepare the double antibody sandwich TRFIA kit following the the optimization of reaction
conditions, and the sensitivity, accuracy, specificity and repeatability of the kit, were evaluated in this study. 3 different types of
clinical stool, vomit, blood samples (60 samples each) of suspected cases and 10 samples from CPV-negative dogs were detected parallelly by the TRFIA kit and RT-PCR method, and their coincidence rate was compared based on the detection results. The final
reaction conditions were optimized and as follows: coating antibody concentration (15 μg/mL); the volumes of detection antibodies
(2.5 mg/mL) and Eu3+ chelates were 70 μL and 30 μL, respectively; the reaction system (25 μL CPV standard or test sample+200
μL analysis buffer+200 μL Eu3+ labeled antibody +200 μL enhancement solution). Sensitivity test showed that the TRFIA method
had a sensitivity of 0.83 ng/mL for the detection of CPV inactivated virus. Accuracy test results showed that the dilution recovery
rate is 89.25%-100.8% at serial dilution (1:2, 1:4 and 1:8); specificity test results showed that the results were all negative when
CDV, CPIV, CAV-1, CCV, FPV, MEV were detected by the test kit without obvious cross-reaction; the results of repeatability test at
high, medium and low dilution of CPVs showed that intra-batch coefficients of variation (CV) is between 2.34% and 4.76%, and
inter-batch CV is between 2.38% and 5.10%; stability test results showed that the kit could be stably stored at 4 ℃ for 6 months
and at 37 ℃ for 7 days; clinical sample test showed that VPV was positive from most of clinical stool, vomit, blood samples of
suspected cases when detected by the TRFIA method, while the relevant samples from the CPV-negative dogs were negative. The
detection results of established TRFIA method were consistent with that of the RT-PCR method, and the coincidence rate reached
100%. The above results showed that the CPV TRFIA kit established in this study had the high sensitivity and accuracy, strong
specificity, good stability and repeatability, and was suitable for the detection of several types of clinical CPV samples. This study
provided an effective technique for the clinical sample detection.

相似文献/References:

[1]陈翠翠,赖宏锐,梁焕坤,等.犬瘟热病毒时间分辨免疫荧光分析方法的建立及初步应用[J].中国预防兽医学报,2020,(03):258.[doi::10.3969/j.issn.1008-0589.201909004]
 CHEN Cui-cui,LAI Hong-rui,LIANG Huan-kun,et al.Establishment and preliminary application of time-resolvedimmunofluorescence analysis method for canine distemper virus[J].Chinese journal of preventive veterinary medicine,2020,(02):258.[doi::10.3969/j.issn.1008-0589.201909004]

更新日期/Last Update: 2021-03-17