Development of a High-Efficiency Immunomagnetic Enrichment Method for Detection of Human Norovirus via PAMAM Dendrimer/SA-Biotin Mediated Cascade-Amplification.

Junshan Gao,Ying Li,Weicheng Cai,Juan Wang,Moutong Chen,Le Zhang,Liang Xue,Yanhui Liang,Jiale Yang,Qingping Wu,Linping Wang,Zhiwei Qin,Qinghua Ye,Jumei Zhang,Shi Wu

doi:10.3389/fmicb.2021.673872

Abstract

Human norovirus is a common cause of acute gastroenteritis worldwide, and oysters have been found to be the main carriers for its spread. The lack of efficient pre-treatment methods has been a major bottleneck limiting the detection of viruses in oysters. In this study, we established a novel immunomagnetic enrichment method using polyamidoamine (PAMAM) dendrimer/SA-biotin-mediated cascade amplification for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. We compared the capture efficiency of traditional immunomagnetic enrichment, biotin-amplified immunomagnetic enrichment, and PAMAM dendrimer/SA-biotin-mediated cascade-amplification immunomagnetic enrichment. The optimal capture efficiency of the novel method was 44.26 ± 1.45%, which increased by 183.17% (P < 0.01) and 18.09% (P < 0.05) compared with the first two methods, respectively. Three methods were all applied in detecting norovirus in 44 retail oysters, the detection rate of the PAMAM dendrimer/SA-biotin-mediated method was 25.0%, which was higher than those of traditional IME (15.90%) and SA-biotin-amplified IME (18.80%) by 9.1 and 6.2%, respectively. In conclusion, the novel method can be applied for the rapid detection of norovirus in oysters, which can help reduce the cost and time of detection and improve detection rates.

Highlights

Norovirus (NoV) is one of the main causes of acute gastroenteritis (Ahmed et al, 2014) and frequently appears in closed spaces in hospitals, schools, and cruise ships (Siebenga et al, 2009)
We aimed to establish a sensitive and efficient PAMAM dendrimer/SA-biotin-mediated cascadeamplification immunomagnetic enrichment (IME) (P-SA-BA-IME) method, which was combined with RT-qPCR to detect NoVs in oysters
The biotin-monoclonal antibody (mAb) was centrifuged in a 30 KDa ultrafiltration tube (Millipore, MWCo, 30000) (Millipore, Carrigtwohill, County Cork, Ireland) at 6,000 × g for 10 min to remove unbound biotin, and washed four times with phosphate buffered saline (PBS) with 0.05% Tween 20 (PBST) (0.01 M, pH 7.4)

Summary

Introduction

Norovirus (NoV) is one of the main causes of acute gastroenteritis (Ahmed et al, 2014) and frequently appears in closed spaces in hospitals, schools, and cruise ships (Siebenga et al, 2009). Real time RT-PCR (RT-qPCR) is the “gold standard” for NoV diagnosis and detection (Vinjé, 2015). This method is often affected by low amounts of NoVs and by presence of inhibitors in the sample, such as polysaccharides, lipids, and proteins in oyster tissue. Traditional enrichment methods include direct treatment, proteinase K digestion and proteinase K digestion combined with polyethylene glycol precipitation (Lee and Lee, 1981; Wu et al, 2016). These methods are non-specific and cannot fully remove the interference of complex matrices

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Conclusion