In situ capture RT-qPCR: a new simple and sensitive method to detect human norovirus in oysters

Authors: ZHOU, ZHENHUAN - Shanghai Jiaotong University; TIAN, ZHENGAN - Shanghai Jiaotong University; LI, QIANQIAN - Shanghai Jiaotong University; Tian, Peng; WU, QINGPING - Guangdong University; WANG, DEPENG - Shanghai Jiaotong University; SHI, XIANMING - Shanghai Jiaotong University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/2017
Publication Date: 4/3/2017
Citation: Zhou, Z., Tian, Z., Li, Q., Tian, P., Wu, Q., Wang, D., Shi, X. 2017. In situ capture RT-qPCR: a new simple and sensitive method to detect human norovirus in oysters. Frontiers in Microbiology. 8:554. https://doi.org/10.3389/fmicb.2017.00554.
DOI: https://doi.org/10.3389/fmicb.2017.00554

Interpretive Summary: Human noroviruses (HuNoVs) are the major cause for the non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to discriminate between infectious and non-infectious viruses. Previously, we developed a receptor mediated capture assay (ISC-RT-qPCR) to concentrate human norovirus and estimate infectivity of HuNoV. In collaboration with scientist from various laboratories in Shanghai, China, we applied this method to detect HuNoV contamination in oysters from local retail markets. We have demonstrated that ISC-RT-qPCR was more sensitive than the widely used RT-qPCR method with a set of clinical samples. In the artificially HuNoV-contaminated oysters, the distribution and the detected viral genomic copy numbers measured by ISC-RT-qPCR were comparable to RT-qPCR. However, the sensitivity of ISC-RT-qPCR was significantly better than RT-qPCR in detection of HuNoV in oysters collected from retail markets. Thirty-six oyster samples from retail markets in Shanghai were detected for HuNoV by both assays. The detection rates of GI HuNoV in gill, digestive glands, and other tissues were 33.3%, 25%, and 19.4% by ISC-RT-qPCR; and were 5.6%, 11.1%, and 11.1% by RT-qPCR. No GII HuNoV was detected in these oysters by both methods. Overall, we demonstrated that ISC-RT-qPCR is a promising method for detecting HuNoVs in clinical and oyster samples.

Technical Abstract: Human noroviruses (HuNoVs) are the major cause for the non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to discriminate between infectious and non-infectious viruses. Previously, we reported that the receptor mediated in situ capture RT-qPCR (ISC-RT-qPCR) could be used as an alternative approach to estimate infectivity of Tulane virus as well as HuNoVs. In this study, we optimized the conditions for ISC-RT-qPCR and applied the method to detect HuNoVs from clinical and oyster samples. Fifteen clinical samples from gastritis patients were used to test sensitivity of ISC-RT-qPCR and compared with RT-qPCR assay. Among 15 clinical samples, 5 samples were known negative for HuNoVs and 10 samples were known positive for HuNoVs determined by RT-PCR and sequencing. All five HuNoV-negative samples were also negative determined by both ISC-RT-qPCR and real-time RT-qPCR. Among 10 HuNoV-positive samples, only 8 samples were positive determined by real-time RT-qPCR and all samples were positive determined by ISC-RT-qPCR. We further demonstrated that the detection limit of ISC-RT-qPCR was better than RT-qPCR. GI HuNoV could be detected at a maximal dilution of 2:106 by RT-qPCR and 2:107 by ISC-RT-qPCR. Similarly, GII HuNoV could be detected at a maximal dilution of 2:106 by RT-qPCR and 2:108 by ISC-RT-qPCR. Thus, ISC-RT-qPCR had at a one-log (GI) or a two-log (GII) increase in the sensitivity than RT-qPCR assay. ISC-RT-qPCR was applied to study distribution of HuNoVs in oysters as well as detection of HuNoVs in oysters from retail market. Thirty oysters were of artificially contaminated with GI or GII HuNoVs. Gills (G), digestive glands (D), and residual tissues (O) were collected and tested by both ISC-RT-qPCR and RT-qPCR. In the artificially HuNoV-contaminated oysters, the distribution and the detected viral genomic copy numbers measured by ISC-RT-qPCR were comparable to RT-qPCR. However, the sensitivity of ISC-RT-qPCR was significantly better than RT-qPCR in detection of HuNoV in oysters collected from retail markets. Thirty-six oyster samples from retail markets in Shanghai were detected for HuNoV by both assays. The detection rates of GI HuNoV in G, D, and O were 33.3%, 25%, and 19.4% by ISC-RT-qPCR; and were 5.6%, 11.1%, and 11.1% by RT-qPCR. No GII HuNoV was detected in these oysters by both methods. Overall, we demonstrated that ISC-RT-qPCR is a promising method for detecting HuNoVs in clinical and oyster samples.