Rapid detection and differentiation of human noroviruses using RT-PCR coupled to electrospray ionization mass spectrometry

• Published in: Food microbiology Vol. 44; pp. 71 - 80
• Main Authors: Hellberg, Rosalee S., Li, Feng, Sampath, Rangarajan, Yasuda, Irene J., Carolan, Heather E., Wolfe, Julia M., Brown, Michael K., Alexander, Richard C., Williams-Hill, Donna M., Martin, William B.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 01-12-2014; Elsevier
• Subjects: Biological and medical sciences; DNA Primers - genetics; Electrospray ionization mass spectrometry; Food Contamination - analysis; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Genotyping; Hepatitis A virus; Humans; Norovirus; Norovirus - chemistry; Norovirus - genetics; Norovirus - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction - methods; RT-PCR; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Norovirus; Hepatitis A virus; Genotyping; Electrospray ionization mass spectrometry; RT-PCR; Human; Picornaviridae; Genotype; Electrospray; Hepatovirus; Virus; Rapid technique; Differentiation; Detection; Reverse transcription polymerase chain reaction; Mass spectrometry
• DOI: 10.1016/j.fm.2014.05.017
• EISSN: 1095-9998
• ISSN: 0740-0020

The goal of this study was to develop an assay for the detection and differentiation of noroviruses using RT-PCR followed by electrospray ionization mass spectrometry (ESI-MS). Detection of hepatitis A virus was also considered. Thirteen primer pairs were designed for use in this assay and a reference database was created using GenBank sequences and reference norovirus samples. The assay was tested for inclusivity and exclusivity using 160 clinical norovirus samples, 3 samples of hepatitis A virus and 3 other closely related viral strains. Results showed that the assay was able to detect norovirus with a sensitivity of 92% and a specificity of 100%. Norovirus identification at the genogroup level was correct for 98% of samples detected by the assay and for 75% of a subset of samples (n = 32) compared at the genotype level. Identification of norovirus genotypes is expected to improve as more reference samples are added to the database. The assay was also capable of detecting and genotyping hepatitis A virus in all 3 samples tested. Overall, the assay developed here allows for detection and differentiation of noroviruses within one working day and may be used as a tool in surveillance efforts or outbreak investigations.
•This paper describes a novel method for the identification of human noroviruses.•RT-PCR was combined with mass spectrometry to develop a foodborne viral assay.•This assay showed a sensitivity of 92% for detection of norovirus in 160 samples.•Genogroup and genotype were correctly identified in the majority of viral samples.•The assay showed 100% specificity and was also able to detect hepatitis A virus.