Design and development of a quantitative real time PCR assay for monitoring of HTLV-1 provirus in whole blood

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• 作者：Mahmood Naderi^a ; ^b ; ^f ; Mahdi Paryan^b ; ^c ; ^f ; Kayhan Azadmanesh^d ; Houshang Rafatpanah^e ; Houri Rezvan^f ; Siamak Mirab Samiee^f ; ^g ; ^{siamak.samiee@gmail.com}
• 关键词：HTLV-1 ; Proviral load ; Quantitative real time PCR ; Tax
• 刊名：Journal of Clinical Virology
• 出版年：2012
• 期刊代码：150_13866532
• 类别：med
• 出版时间：April, 2012
• 卷：53
• 期：4
• 页码：302-307
• 文件大小：1136 K

摘要

Background

Proviral load quantification of human T-lymphotropic virus type 1 (HTLV-1) is an essential marker for disease progression. Therefore, accurate and precise quantification of the virus is important. However, many articles published about detection and quantification of HTLV-1 virus neither reported any databank for the pre-validation of their primer and probe sequences nor stressed on its importance. Consequently, this failure may cause proviral load measurement variations of different HTLV-1 strains.

Objective

The aim of this study was to develop a TaqMan assay for HTLV-1 proviral load quantification which is based on a conserved region of tax gene with minimal sequence variability.

Study design

For the purpose of finding the most conserved region of tax gene, all the HTLV-1 Gene Bank records including tax gene sequence (524 records by December 2009) were aligned in order to design on the most conserved region of this gene. The specificity, sensitivity, inter and intra assay and the dynamic range of the assay were experimentally determined by their respective methodology.

Result

The assay has a dynamic range of 10-10⁷ HTLV-1 plasmid DNA/rxn (reaction) and the limit of detection (LOD) less than 10 copies/rxn. The assay gave coefficient of variation (CV) for the Ct values of less than 1%and 4.8%for intra and inter assay, respectively. Clinical sensitivity and specificity were determined to be 97.8%and 100%, respectively.

Conclusion

This TaqMan assay is able to reliably quantify proviral load due to the fact that it has been designed on a conserved region of HTLV-1 tax gene with minimal sequence variability.