An optimized nested polymerase chain reaction (PCR) approach allows detection and characterization of human immunodeficiency virus type 1 (HIV-1) env and gag genes from clinical samples.
The needs for development and/or improvement of molecular approaches for microorganism detection and characterization such as polymerase chain reaction (PCR) are of high interest due their sensitivity and specificity when compared to traditional microbiological techniques. Considering the worldwide importance of human immunodeficiency virus type 1 (HIV-1) infection, it is essential that such approaches consider the genetic variability of the virus, the heterogeneous nature of the clinical samples, the existence of contaminants and inhibitors, and the consequent needs for standardization in order to guarantee the reproducibility of the methods. In this work we describe a nested PCR assay targeting HIV-1 virus gag and env genes, allowing specific and sensitive diagnosis and further direct characterization of clinical samples. The method described herein was tested on clinical samples and allowed the detection of HIV-1 presence in all samples tested for the gag gene and 90.9% for the env gene, revealing sensitivities of 1 fg and 100 fg, respectively. Also, no cross-reactions were observed with DNA from infected and noninfected patients and the method allowed detection of the env and gag genes on an excess of 10(8) and 10(4) of human deoxyribonucleic acid (DNA), respectively. Furthermore, it was possible to direct sequence all amplified products, which allowed the sub typing of the virus in clinical samples.
Locateli
Dayse
D
Laboratório de Imunologia Aplicada, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
Stoco
Patricia H
PH
Zanetti
Carlos R
CR
Pinto
Aguinaldo R
AR
Grisard
Edmundo C
EC
D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D015686:Gene Products, env / Q000032:analysis* / Q000235:genetics*; D015683:Gene Products, gag / Q000032:analysis* / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015686:Gene Products, env; D015683:Gene Products, gag; D004996:Ethidium