A simple and rapid genotyping method for beta-2 receptor (beta2 AR) gene using allele specific multiplex PCR.

BACKGROUND: Polymorphism of the beta2-adrenergic receptor (beta2 AR) gene is an important determinant of the function of this receptor. It affects receptor down-regulation and beta2-agonist responses. It has also been a focus of interest in attempts to elucidate the genetic basis of asthma, hypertension, obesity and cystic fibrosis. Several different techniques have been established to determine beta2 AR genotypes but none of these methods are simple enough to detect simultaneously all the five alleles of our research interest (Arg16/Gly16, -20T/C, Gln27/Glu27, -47T/C and Thr164/Ile164). OBJECTIVES: To develop a simple and rapid PCR based method for the simultaneous detection of five beta2 AR alleles. METHOD: DNA was extracted from whole blood using standard alkali lysis method. Primers specific at the 3' end for the polymorphic sites were designed. The nested allele specific PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, Taq polymerase, primers and annealing temperature, to produce specific bands of interest. DNA samples were selected at random and submitted for direct PCR sequencing. RESULT: The first PCR produced a fragment of size 710 bp, which was used as template for the subsequent duplex and triplex PCR to detect the mutation sites of the five alleles. The method was found reproducible and specific when used to genotype patients with bronchial asthma. The sequencing results confirmed the specificity of the PCR method. CONCLUSION: The simple and rapid method for the simultaneous detection of the five beta2 AR alleles is suitable for the study of beta2 polymorphism and its clinical consequences.

Comparative results in detection of HCV antibodies by using a rapid HCV test, ELISA and immunoblot.

Viral hepatitis C is a major problem of post-transfusion hepatitis. The best measure for preventing hepatitis C infection in transfusion is blood donor screening. There are many methods for detecting hepatitis C virus antibodies. ELISA is one of the sensitive screening methods. However it needs equipment, technical skills and it is time consuming. The Genelabs Diagnostics HCV-SPOT is a simple, rapid test for the detection of anti-HCV. This paper reports comparative results in detection of HCV antibodies by using GLD HCV-SPOT, ELISA and GLD/DBL HCV BLOT. One hundred and ninety-two specimens from blood donors patients with chronic hepatitis, cirrhosis, hepatoma and miscellaneous chronic liver diseases were tested by HCV-spot assay and HCV ELISA. The positive specimens were confirmed by the immunoblot assay. Only one of 140 samples negative by HCV-SPOT was positive by ELISA. The sensitivity and specificity of HCV-SPOT assays were 97.6% and 92.6% respectively. The positive predictive value was 78.8%, negative predictive value was 99.3%, accuracy was 93.6%. The rapid HCV-SPOT assay can be used in the management of transplantation graft procedures or emergency blood screening for transfusion. In addition, it can be used in small, local hospitals without any expensive equipment.