Detection of severe acute respiratory syndrome probable patients' virus RNA in Hangzhou by using a two loci and a modified nested real-time reverse transcription polymerase chain reaction.
- Author:
Rong YE
1
;
Jin-cao PAN
;
Zhi-cheng HUANG
;
Heng WANG
;
Hao-qiu WANG
;
Dong-fang WEI
;
Ke XU
;
Hong-gen WEN
;
Kang-kai CHEN
Author Information
- Publication Type:Journal Article
- MeSH: Adolescent; Adult; Aged; Aged, 80 and over; Base Sequence; Humans; Middle Aged; RNA, Viral; genetics; metabolism; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; methods; SARS Virus; genetics; Sensitivity and Specificity; Severe Acute Respiratory Syndrome; diagnosis; virology; Young Adult
- From: Chinese Journal of Preventive Medicine 2005;39(2):129-132
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo detect the RNA of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcription polymerase chain reaction (RT-PCR) targeted for a two loci and a modified nested real-time RT-PCR as to improving the reliability and sensitivity of tests.
METHODSA nested RT-PCR was used for detecting one fragment of SARS-CoV RNA in oropharyngeal swabs from 3 SARS probable patients, 4 SARS suspect patients and other 27 patients with fever in Hangzhou, and the nested RT-PCR product from one SARS probable patient was sequenced. Meanwhile in these 3 SARS probable patients, other three RT-PCR methods, including a hemi-nested RT-PCR targeted for another fragment of SARS-CoV RNA, a real-time RT-PCR and a modified nested real-time RT-PCR, were employed to detect SARS-CoV RNA.
RESULTSTwo positives were found in the 3 SARS probable patients, and none positive in 4 SARS suspect patients and other 27 patients with fever, using the nested RT-PCR. The sequence of the nested RT-PCR product from one SARS probable patient was identified with the counterpart of SARS-CoV genomes published in public database. The results of the hemi-nested RT-PCR, the real-time RT-PCR and the modified nested real-time RT-PCR in the 3 SARS patients were consistent with the one of the nested RT-PCR. During detecting specimen with low copies of RNA, a weak positive signal was produced after about 35 cycles in the real-time RT-PCR, but a strong positive signal was found only after 10 cycles in the modified nested real-time RT-PCR.
CONCLUSIONIt might improve the reliability of test by employing RT-PCR targeted for two or more fragments in SARS-CoV genome. The modified nested real-time RT-PCR might have higher sensitivity than the routine real-time RT-PCR.