Simplified PCR detection method for nasal Mycobacterium leprae.
We report here a simplified method for the detection of nasal carriage of Mycobacterium leprae. DNA extracted from nasal swabs was analyzed by PCR, and M. leprae specific amplicons detected by means of a novel peptide-nucleic-acid-ELISA (PNA-ELISA) method. Parameters for the method were established using swabs taken from untreated lepromatous leprosy patients. We have developed this method to study nasal carriage in endemic populations. However, due to the sensitivity of PCR based techniques, we wished to assess the possibility of false positive samples arising in our method. We therefore examined samples taken from individuals in Norway, a country non-endemic for leprosy, using our technique. A total of 219 nasal swabs were collected and tested in our laboratory in London. All of these were found to be negative by our criteria. In order to corroborate our results, and also to assess the specificity of the method, a small number of these samples were randomly selected, and a known amount of M. leprae DNA added to them. All 219 samples were then retested using the same techniques under "double blind" conditions in our laboratory in India. All of the samples to which M. leprae DNA had been added were successfully identified by this method whereas all other swabs were negative. Taken together, these results suggest that the technique described here is simple, sensitive, and specific for use in large-scale epidemiological studies. This study, part of the larger MILEP 2 study, represents the first use of a PNA-PCR method for an epidemiological study of infection. The method using PNA-ELISA is significantly simpler and more rapid than gel based detection methods. The supply of laboratory consumables and overall detection procedure were simplified and standardized by use of PCR Ready-to-Go beads.
Jadhav
R S
RS
Richardson Leprosy Hospital, Sangli Road, Miraj, India 416410.
Macdonald
M
M
Bjune
G
G
Oskam
L
L
D004269:DNA, Bacterial / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007918:Leprosy / Q000382:microbiology*; D009166:Mycobacterium leprae / Q000235:genetics / Q000302:isolation & purification*; D009666:Nose / Q000382:microbiology*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020135:Peptide Nucleic Acids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D020135:Peptide Nucleic Acids