目的:犬肠道冠状病毒(CCV)和犬细小病毒2型(CPV-2)是导致犬急性胃肠炎的主要病原体,单一和混合感染都很常见。本研究旨在建立双标记时间分辨荧光免疫测定法(TRFIA),以检测和区分CCV和CPV-2疾病。
方法:采用铕(Ⅲ)(Eu3+)/钐(Ⅲ)(Sm3+)螯合物建立并优化了夹心双标记TRFIA方法。CCV/CPV-2抗原首先被固定的抗体捕获。然后,结合Eu3+/Sm3+标记的配对抗体,解离后检测Eu3+/Sm3+荧光值,计算CCV/CPV-2比值。表演,用于实验室的临床表现和方法学(灵敏度,特异性,准确性和稳定性)测试进行了评估。
结果:优化并建立了用于CCV和CPV-2检测的双标记TRFIA。此TRFIA试剂盒对CCV的灵敏度为0.51ng/mL,对CPV-2的灵敏度为0.80ng/mL,对CCV和CPV-2具有高特异性。所有精度数据均小于10%,回收率在101.21至110.28%之间。试剂盒可以在4°C下暂时储存20天,并且可以在低于-20°C的温度下储存12个月。根据对137名临床可疑患者的方法学比较,TRFIA试剂盒与PCR法比较差异无统计学意义。此外,对于CCV检测,临床敏感性为95.74%,临床特异性为93.33%。对于CPV-2检测,临床敏感性为92.86%,临床特异性为96.97%。
结论:在这项研究中,制备了用于CCV和CPV-2检测的双标记TRFIA试剂盒,具有较高的实验室灵敏度,特异性,准确度,稳定性,临床敏感性和特异性。该试剂盒为筛选/区分CCV和CPV-2提供了新的选择,并可能有助于改进将来预防和控制动物传染病的策略。
Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases.
A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated.
A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than - 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%.
In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.