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. 2025 Mar 21;13(4):712.
doi: 10.3390/microorganisms13040712.

Evaluation and Application of the MIRA-qPCR Method for Rapid Detection of Norovirus Genogroup II in Shellfish

Yanting Zhu  1 , Mengyuan Song  1 , Yingjie Pan  1   2   3 , Yong Zhao  1   2   3 , Haiquan Liu  1   2   3   4
Affiliations

Evaluation and Application of the MIRA-qPCR Method for Rapid Detection of Norovirus Genogroup II in Shellfish

Yanting Zhu et al. Microorganisms. .

Abstract

Globally, norovirus has become the primary cause of outbreaks of acute gastroenteritis, and an increasing number of norovirus GII infections have been associated with shellfish. This highlights the urgent need to establish sensitive and rapid detection platforms for timely screening of contaminated shellfish to reduce the risk of virus transmission. To address this challenge, we developed a novel detection method combining multienzyme isothermal rapid amplification (MIRA) with qPCR, referred to as MIRA-qPCR, specifically targeting norovirus GII. It exhibited robust specificity, demonstrating no cross-reactivity with sapovirus, rotavirus, hepatitis A virus, Escherichia coli, Listeria monocytogenes, or Vibrio parahaemolyticus, and exhibited high sensitivity, detecting as low as 1.62 copies/μL for recombinant plasmid standards. Furthermore, MIRA-qPCR showed good linearity in the 1.62 × 101 to 1.62 × 107 copies/μL range, with an R2 > 0.90. MIRA-qPCR and qPCR assays were performed on 125 fresh shellfish samples; there was good consistency in the detection results, and the Kappa value was 0.90 (p < 0.001). The sensitivity and specificity of the MIRA-qPCR detection were 100.00% and 97.25%, respectively. The MIRA-qPCR technique provides a viable alternative for the rapid screening of norovirus GII-contaminated shellfish to guarantee food safety.

Keywords: MIRA–qPCR; food safety; norovirus GII; rapid amplification; shellfish.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic diagram of the process of rapid detection of norovirus GII using MIRA–qPCR technology. The detection process is divided into three steps: (A) RNA extraction and reverse transcription, created with MedPeer.com. (B) Gene amplification and fluorescence signal acquisition by MIRA–qPCR, i.e., a. the Rec/SSDNA complex is formed; b. invasion of the template and forming of a D-loop region; c. DNA strand extension; d. probe hybridization; e. release of fluorescent groups. (C) Curve amplification obtained and results read.
Figure 2
Figure 2
Optimization of reaction conditions. (A) 2% gel plot for basic MIRA screening primers. M: D2000 marker—lane 1: Nf1/NR1; lane 2: Nf1/NR2; lane 3: Nf1/NR3; lane 4: Nf2/NR1; lane 5: Nf2/NR2; lane 6: Nf2/NR3; lane 7: Nf3/NR2; lane 8: Nf3/NR3; lane 9: Nf3/NR1. (B) MIRA–qPCR temperature optimization: 105 copies/μL were selected and amplified at 38, 39, 40, 41, and 42 °C for 20 min. NC: negative control.
Figure 3
Figure 3
Specificity analysis. (A) a: 105 copies/μL (norovirus GII); b: 105 copies/μL (SV); c: 105 copies/μL (HAV); d: 105 copies/μL (RV); e: 105 CFU/mL (E. coli); f: 105 CFU/mL (LM); g: 105 CFU/mL (VP); h: negative control. (B) The Ct plot was calculated to correspond to the amplification plot shown in (A).
Figure 4
Figure 4
The LOD-relevant results of norovirus GII using MIRA–qPCR and conventional PCR. (A) The amplification curves show the LOD of MIRA–qPCR. NC: negative control. (B) The Ct plot was calculated to correspond to the amplification plot shown in (A). (C) The linear regression standard curve between the Ct values and the logarithm concentrations of norovirus GII, as well as the 95% confidence band, were obtained using the software Origin 2021. (D) The LOD of conventional PCR. M: D2000 Marker, lanes 0–7; the corresponding concentrations of norovirus GII were 1.62 × 100~107 copies/μL; lane 8: negative control.
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