Enhancement of detection and quantification of rotavirus in stool using a modified real-time RT-PCR assay

被引:114
作者
Freeman, Molly M.
Kerin, Tara
Hull, Jennifer
McCaustland, Karen [1 ,2 ]
Gentsch, Jon
机构
[1] Natl Ctr Immunizat & Resp Dis, Ctr Dis Control & Prevent, Atlanta, GA USA
[2] Natl Ctr Preparedness, Detect & Control Infect Dis, Ctr Dis Control & Prevent, Atlanta, GA USA
关键词
rotavirus; real-time RT-PCR; NSP-3; children; diarrhea;
D O I
10.1002/jmv.21228
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A sensitive and specific real-time RT-PCR assay to detect rotavirus in stool samples was optimized and validated using a wide range of rotavirus genotypes. The target of the original TaqMan (R) assay is an 87 bp fragment of the highly conserved non-structural protein 3 (NSP3) gene. Here we modified the original assay by introducing degeneracy into the forward primer to account for sequence variation between rotavirus genotypes, added four nucleotides at the Tend of the reverse primer to reduce its stability, and modified the probe label. Amplification and detection conditions were optimized using purified dsRNA from two cultivated strains. The limit of detection of the modified assay was calculated to be approximately 44 genome copies per reaction. To validate the reactivity of the assay, 103 archived RNAs that had been extracted from stools and genotyped during routine U.S. surveillance were tested. Samples were selected to represent both rare and common genotypes that have been detected in U.S. children. Nine genotypes known to be circulating in the United States were detected by the real-time assay demonstrating broad reactivity. In addition, other enteric viruses were not detected demonstrating that the assay is specific for rotavirus and does not cross-react with other viruses potentially present in stool samples. This real-time assay is an important addition to the arsenal of molecular tools available to quickly identify rotavirus in stool samples during routine surveillance.
引用
收藏
页码:1489 / 1496
页数:8
相关论文
共 36 条
[1]   Basic principles of real-time quantitative PCR [J].
Arya, M ;
Shergill, IS ;
Williamson, M ;
Gommersall, L ;
Arya, N ;
Patel, HRH .
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2005, 5 (02) :209-219
[2]   Excretion of serotype G1 rotavirus strains by asymptomatic staff: A possible source of nosocomial infection [J].
Barnes, GL ;
Callaghan, SL ;
Kirkwood, CD ;
Bogdanovic-Sakran, N ;
Johnston, LJ ;
Bishop, RF .
JOURNAL OF PEDIATRICS, 2003, 142 (06) :722-725
[3]   Rotavirus antigenemia in children is associated with viremia [J].
Blutt, Sarah E. ;
Matson, David O. ;
Crawford, Sue E. ;
Staat, Mary Allen ;
Azimi, Parvin ;
Bennett, Berkeley L. ;
Piedra, Pedro A. ;
Conner, Margaret E. .
PLOS MEDICINE, 2007, 4 (04) :660-668
[4]   Rotavirus antigenaemia and viraemia:: a common event? [J].
Blutt, SE ;
Kirkwood, CD ;
Parreño, V ;
Warfield, KL ;
Ciarlet, M ;
Estes, MK ;
Bok, K ;
Bishop, RF ;
Conner, ME .
LANCET, 2003, 362 (9394) :1445-1449
[5]   RAPID AND SIMPLE METHOD FOR PURIFICATION OF NUCLEIC-ACIDS [J].
BOOM, R ;
SOL, CJA ;
SALIMANS, MMM ;
JANSEN, CL ;
WERTHEIMVANDILLEN, PME ;
VANDERNOORDAA, J .
JOURNAL OF CLINICAL MICROBIOLOGY, 1990, 28 (03) :495-503
[6]   Quantitative real-time RT-PCR - a perspective [J].
Bustin, SA ;
Benes, V ;
Nolan, T ;
Pfaffl, MW .
JOURNAL OF MOLECULAR ENDOCRINOLOGY, 2005, 34 (03) :597-601
[7]   CHARACTERIZATION OF ROTAVIRUS STRAINS FROM NEWBORNS IN NEW-DELHI, INDIA [J].
DAS, BK ;
GENTSCH, JR ;
CICIRELLO, HG ;
WOODS, PA ;
GUPTA, A ;
RAMACHANDRAN, M ;
KUMAR, R ;
BHAN, MK ;
GLASS, RI .
JOURNAL OF CLINICAL MICROBIOLOGY, 1994, 32 (07) :1820-1822
[8]   Symptomatic and subclinical infection with rotavirus P[8]G9, rural Ecuador [J].
Endara, Pablo ;
Trueba, Gabriel ;
Solberg, Owen D. ;
Bates, Sarah J. ;
Ponce, Karina ;
Cevallos, William ;
Matthijnssens, Jelle ;
Eisenberg, Joseph N. S. .
EMERGING INFECTIOUS DISEASES, 2007, 13 (04) :574-580
[9]  
Estes MK., 2007, FIELDS VIROLOGY, P1917
[10]   Hospitalizations and deaths from diarrhea and rotavirus among children <5 years of age in the United States, 1993-2003 [J].
Fischer, Thea Kolsen ;
Viboud, Cecile ;
Parashar, Umesh ;
Malek, Mark ;
Steiner, Claudia ;
Glass, Roger ;
Simonsen, Lone .
JOURNAL OF INFECTIOUS DISEASES, 2007, 195 (08) :1117-1125