Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6).
HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/microL (equivalent to 2465.8 molecules/microL) to 10-9 (equivalent to 2.46 molecules/microL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/microL and for the nested PCR was 2.46 molecules/microL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.
Canto
Cynthia Liliane Motta do
CL
Laboratório de Virologia, Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP, Brazil. ccanto@usp.br
Sumita
Laura Massami
LM
Machado
Adriana Freire
AF
Tateno
Adriana
A
Cunha
Eveline Vieira da
EV
Machado
Clarisse Martins
CM
D052160:Benzothiazoles; D004279:DNA, Viral / Q000032:analysis; D003959:Diamines; D005077:Exanthema Subitum / Q000175:diagnosis*; D005456:Fluorescent Dyes; D015654:Herpesvirus 6, Human / Q000235:genetics*; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I