Optimization of the Sybr Green Real Time PCR for the detection of human herpes virus type 6 (HHV-6)

作者：Motta do Canto Cynthia Liliane; Sumita Laura Massami; Machado Adriana Freire; Tateno Adriana; da Cunha Eveline Vieira; Machado Clarisse Martins

来源：Revista do Instituto de Medicina Tropical de Sao Paulo, 2008, 50(1): 61-63.

DOI：10.1590/s0036-46652008000100014

• human herpes virus 6 (HHV-6)
• PCR
• real-time PCR
• Sybr Green

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 Intent 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/mu L (equivalent to 2465.8 molecules/mu L) to 10-(9) (equivalent to 2.46 molecules/mu L). 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/mu L and for the nested PCR was 2.46 molecules/mu L. 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.

• 出版日期2008-2
• 单位河北医科大学