# Advances and Future Perspective on Detection Technology of Human Norovirus
## 2 Detection Techniques
### Molecular Detection Methods
#### 2.3.2 Thermal Cycling Amplification  
RT-PCR is the most widely used molecular detection technique because of its strong specificity and high sensitivity. The first RT-PCR detection was developed for the relatively conserved RNA polymerase gene in ORF1 [59]. However, primers designed for RT-PCR could not meet the genetic diversity of HuNoVs. In addition, RT-PCR has been applied in the ORF1-ORF2 junction region for the HuNoV genome (the most conserved region). However, RT-PCR requires high-quality RNA templates, and it is a time-consuming process and has high sensitivity to RNA inhibitors. Therefore, RT-qPCR was explored. Given its high sensitivity and specificity, RT-qPCR is currently known as the standard for the detection of HuNoVs in food and environmental samples. [PERSON] et al. reported the first set of primer probes for the detection of GI and GII HuNoVs and established relative RT-qPCR [60]. Moreover, [PERSON] et al. established one-step TaqMan probe RT-qPCR to detect GI and GII HuNoVs in shellfish and clinical samples [61]. The method took 90 min, and the sensitivity increased by 10-100 times compared with the traditional one-step RT-PCR. [PERSON] et al. updated the primers and probes and designed dual RT-qPCR todetect GI and GII HuNoVs, simultaneously [62]. Compared with the previously reported RT-qPCR, dual RT-qPCR had higher specificity and sensitivity with a processing time of 40 min. This method not only ensured the reliability of the results but also improved efficiency during detection for a large quantity of samples. Virions have potential infectivity. However, RT-PCR and RT-qPCR, based on the detection of viral RNA, cannot distinguish infectious virions, inactive virions, or free RNA. It may result in misjudgment of the infection. In addressing the problem, [PERSON] et al. used antibodies (PAbs for VLPs) and magnetic beads to capture viral particles from stool samples [63]. This method could remove inhibitors effectively and improve the accuracy of the detection. However, the HuNoV genotypes that can be detected by this method were limited. Afterwards, detection methods based on magnetic bead HBGAs and magnetic bead PGM were successively reported to detect HuNoVs particles instead of free RNA [64; 65; 66]. [PERSON] et al. developed ISC-RT-qPCR using HBGAs to capture viral particles [67]. This method could evaluate the inactivation effect of HuNoVs by disinfection methods such as heating, chlorine and UV [68]. It was also used to detect potentially infectious HuNoVs in environmental water samples [69], clinical samples and commercial oyster samples [70]. The results showed that compared with traditional RT-qPCR, this method had higher sensitivity, particularly for potentially infectious HuNoVs. ISC-RT-qPCR simplified the steps for virion concentration and viral RNA extraction and eliminated the effects of PCR inhibitors and free RNA of HuNoVs. Therefore, ISC-RT-qPCR had great advantages in food, environmental and clinical sample detection. New PCR-based detection techniques have been reported in recent years. [PERSON] et al. developed a HRPzyme-PCR colorimetric method for HuNoV detection [71]. This method connected HRPzyme onto PCR primers. When a substrate was added into a PCR product, the change in colour caused by the HRPzyme reaction was recorded by a microplate reader. For the detection of GI and GII HuNoV in oyster samples, the recovery rates ranged from 92% to 105% by this method. Moreover, RT-qPCR was developed and widely used, which was suitable for samples with complex compositions and low viral loads. In this method, microfluidization or microdroplets are adopted to disperse the diluted nucleic acid solution into hundreds or even millions of microreactors or droplets on a chip. Then, the number of nucleic acid templates in each microreactor or droplet is less than or equal to one. After PCR, a microreactor or droplet containing the nucleic acid template will have a fluorescence signal. On the contrary, microreactor or droplet without the template will have no signal.