Comparison of microplate hybridization with gel electrophoresis and dot blot hybridization for the rapid detection of Mycobacterium tuberculosis PCR products.
A microplate ELISA hybridization assay has been developed for the detection of the IS6110 PCR products of M. tuberculosis from sputum specimens. In this study, its efficacy was evaluated by comparison with agarose gel electrophoresis (AGE) and dot blot hybridization (DBH), with culture results as the 'gold standard'. The assay was used with 190 sputum samples: the PCR results detected by ELISA and AGE showed close agreement, with sensitivity, specificity and accuracy of 90%, 100% and 96% respectively. The same values for DBH were 92%, 98% and 96% respectively. The validities of these methods were not statistically significantly different (p>0.05). The agreement rates of PCR product detection by AGE comparing with DBH and ELISA were 0.964 and 0.964 respectively, while that of DBH and ELISA was 1.0 by Kappa analysis. The overall agreement was not statistically significantly different (p>0.05). Use of DBH or ELISA hybridization increased the sensitivity of detection by AGE 10-fold from 10 pg to 1 pg of purified DNA per reaction; ie from about 30 to about 3 organisms. The amount of PCR product detected by ELISA was only one half of that detected by the other methods; the total assay time of ELISA following the PCR was 4 hours. In conclusion, the microplate hybridization assay may replace AGE and DBH for the detection of the PCR products of M. tuberculosis because of its sensitivity, specificity and accuracy. Additional advantages of the microplate assay over AGE and DBH include rapidity, ease of use, greater safety, cost effectiveness and greater objectivity in the reading of results; the technique is suitable for use in epidemiological studies for the analysis of a large number of samples.
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand. unchalee@loxley.co.th
Unchalee
U
Tansuphasiri
Sarawut
S
Suttirat
Somsak
S
Rienthong
D001483:Base Sequence; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013785:Thailand
D017931:DNA Primers