key: cord-0034582-rhy380fw
authors: Rockett, Rebecca
title: Human Coronaviruses
date: 2010-03-27
journal: PCR for Clinical Microbiology
DOI: 10.1007/978-90-481-9039-3_42
sha: 7c89b5bca058bd5f9ba63674b32102a9847c8d75
doc_id: 34582
cord_uid: rhy380fw

Human Coronaviruses (HCoVs) are recognised to be an important cause of the common cold. In 1962 HCoV-229E and HCoV-OC43 where first recognised, more recently HCoV-NL63 and HCoV-HKU-1 have been discovered in respiratory specimens from children and adults [3]. This protocol describes two real-time reverse-transcriptase polymerase chain reaction (RT-PCR) methods, a single target and triplex RT-PCR that identifies and differentiates HCoV infection.

Replicase 1b (2) HCoV-HKU-1-F CCTTGCGAATGAATGTGCT HCoV-HKU-1-R TTGCATCACCACTGCTAGTACCAC HCoV-HKU-1-R FAM-TGTGTGGCGGTTGCTATTATGTTAAGCCTG-BHQ-1

HCoV-229E-F CAGTCAAATGGGCTGATGCA HCoV-229E-R AAAGGGCTATAAAGAGAATAAGGTATTCT N gene (1) HCoV-229E-Pr FAM-CCCTGACGACCACGTTGTGGTTCA-BHQ HCoV-OC43-F CGATGAGGCTATTCCGACTAGGT HCoV-OC43-R CCTTCCTGAGCCTTCAATATAGTAACC N gene (1) HCoV-OC43-Pr QUASAR-TCCGCCTGGCACGGTACTCCCT-BHQ HCoV-NL63-F ACGTACTTCTATTATGAAGCATGATATTAA HCoV-NL63-R AGCAGATCTAATGTTATACTTAAAACTACG 1a gene (3) HCoV-NL63-Pr VIC-ATTGCCAAGGCTCCTAAACGTACAGGTGTT- TAMRA (300)

The first assay contains a single target for HCoV-HKU1, the conserved primer and probe sequences are found in the replicase 1b gene. The second assay detects three HCoV strains, HCoV-229E, HCoV-OC43 both contain target sequences in the N gene, and HCoV-NL63 which target sequences are in the 1a gene (Table 42. (Table 42 .1), in a total reaction volume of 25 μl including 5 μl of sample RNA. Both amplification reactions were performed on the RotorGene 3000 or 6000 (QIAGEN, Australia) using the following parameters; initial RT incubation of 20 min at 50 • C, followed by 50 cycles of 95 • C for 15 s, and 60 • C for 1 min, with fluorescence acquired at the end of each 60 • C step.

The single target HCoV-HKU1 assay was validated using 31 clinical specimens positive for HCoV, detected using various different techniques. No cross reaction was detected to samples positive for other respiratory pathogens. Sensitivity was measured by serial titration, 15 replicates where tested with 100% detection at 50 copies and 33.3% at 5 copies [1] . The HCoV triplex was validated by ATCC positive specimens for each strain. Each positive was tested as an individually target and in the triplex reaction with no loss of sensitivity or cross reaction between strains [2] .

Human coronavirus infections in rural Thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays

Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions

Frequent Detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction