title: A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus.
abstract: A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.
authors.affiliation: Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wei
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.forename: Congliang
authors.initials: C
authors.lastname: Deng
authors.forename: Chengzhong
authors.initials: C
authors.lastname: Zheng
authors.forename: Ximeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Dan
authors.initials: D
authors.lastname: Ma
authors.forename: Yong
authors.initials: Y
authors.lastname: Yi
mesh_terms: D019350:Astroviridae Infections / Q000175:diagnosis* / Q000821:virology; D002675:Child, Preschool; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D001257:Mamastrovirus / Q000302:isolation & purification*; D046529:Microscopy, Electron, Transmission; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*