Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus.
A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10(7) PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.
Shu
Pei-Yun
PY
Division of Research Development and Laboratory Diagnosis, Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China.
Chang
Shu-Fen
SF
Kuo
Yu-Chung
YC
Yueh
Yi-Yun
YY
Chien
Li-Jung
LJ
Sue
Chien-Lin
CL
Lin
Ting-Hsiang
TH
Huang
Jyh-Hsiung
JH
D000208:Acute Disease; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D052160:Benzothiazoles; D002460:Cell Line; D017931:DNA Primers; D003715:Dengue / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000254:growth & development / Q000302:isolation & purification; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D013045:Species Specificity
D000914:Antibodies, Viral; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I