[Detection of human rhinovirus genes from clinical sample by one-step RT-PCR].
OBJECTIVE
Human rhinovirus (HRV) is the most common respiratory pathogen, which causes not only acute respiratory infection and community acquired pneumonitis in children, but also asthma episode and deterioration. However, the detection of respiratory pathogen, which mainly focuses on respiratory syncytial virus, influenzaviruses A and B, parainfluenza viruses 1-3 and adenoviruses, does not include HRV yet by now in China. The absence of detection method limits the clinical understanding of HRV pathogenicity, and causes unreasonable use of antibiotics. This study aimed to establish a one-step reverse transcription (RT) PCR system for detecting specific fragment of HRV RNA, and to analyze the sequences of amplicons.


METHODS
A pair of degenerate primers based on the HRV highly conserved 5'' noncoding region (NCR) were used to develop a one-step RT-PCR system for detecting HRV RNA in nasopharyngeal aspirates from 78 children with acute respiratory tract infections in the spring of 2004. All the positive PCR products were sequenced, and the sequences of the nucleotides were analyzed by using biological software and compared with those in the GeneBank.


RESULTS
Eleven (14.1%) of 78 samples were positive on RT-PCR, these patients were clinically diagnozed as upper respiratory tract infection (n = 7), bronchitis (n = 3) and bronchopneumonia (n = 1), respectively. Compared with the sequences of clinical and standard HRV viruses in the GeneBank, the nucleotide sequences of these 11 amplicons shared high homology of 89%-95.5%. Within the 11 amplicons, nucleotide identity varied from 75.2% to 91.8%, and the ratio of genetic variation was from 8.8% to 31.0%, which occurred in highly conserved regions and usually showed single nucleotide mutation in some special locations. These 11 amplicons attribute to the two branches of HRV cladogram, respectively. Most of mutations in highly conservative domain occurred on single ribonucleotide, mainly as transversion (C/G, A/G) and transition (T/C, A/G), some were mutations among 3 bases (A/C/G, A/T/G, A/C/T). And a few mutations involved two nearby ribonucleotide which were also found in highly conservative domain. However, ribonucleotide deletion and insertion were usually found in highly variable domain.


CONCLUSIONS
The findings showed that this one-step RT-PCR system was highly specific, rapid and convenient for the detection of HRV RNA in nasopharyngeal secretions of patients with acute respiratory tract infections and that the genome of HRV viruses was highly variable.
Xiang
Zhi-dan
ZD
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Fang
Feng
F
Dong
Yong-sui
YS
Cui
Wen
W
Li
Ge
G
D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D005260:Female; D005814:Genes, Viral; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010850:Picornaviridae Infections / Q000175:diagnosis* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D012141:Respiratory Tract Infections / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012229:Rhinovirus / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D012367:RNA, Viral