A simplified reverse transcriptase PCR for rapid detection of Mycobacterium leprae in skin specimens.
An RNA-based assay is an additional molecular tool for leprosy diagnosis and determination of the viability of leprosy bacilli. To simplify RNA detection, a one-step reverse transcriptase PCR (RT-PCR) was established and evaluated. RNA and DNA could be isolated simultaneously. With the use of Mycobacterium leprae-specific primers targeting a 171-bp fragment of the M. leprae 16S RNA gene, RT-PCR resulted in detection of M. leprae in both slit skin smears and skin biopsy specimens. To enhance the positive signal, a digoxigenin-labeled DNA was developed, and successfully detected the amplified RT-PCR product. The method is sensitive, as it could detect one leprosy bacillus. When it was used directly on skin specimens collected from leprosy patients, 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases showed positive results. The assay was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the multidrug therapy for 6 months, resulting in bacterial clearance, 16 of 36 MB patients and three of 24 PB patients tested were still positive for the 16S rRNA gene of M. leprae, suggesting the advisability of a more prolonged treatment course. This form of RT-PCR is of value in terms of simplicity and sensitivity in identifying M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.
Phetsuksiri
Benjawan
B
Sasakawa Research Building, Thai National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand.
Rudeeaneksin
Janisara
J
Supapkul
Prapaporn
P
Wachapong
Suksawat
S
Mahotarn
Krisada
K
Brennan
Patrick J
PJ
D001706:Biopsy; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D007918:Leprosy / Q000175:diagnosis* / Q000235:genetics; D009166:Mycobacterium leprae / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017192:Skin Diseases, Bacterial / Q000175:diagnosis* / Q000235:genetics
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S