Methods in pathology. An improved method for DNA diagnosis of leprosy using formaldehyde-fixed, paraffin-embedded skin biopsies.
To improve the sensitivity of the previously reported polymerase chain reaction (PCR) for the detection of Mycobacterium (M.) leprae in the formaldehyde-fixed, paraffin-embedded tissues, we adapted the PCR designed to amplify an internal 372 bp fragment of a M. leprae-specific repetitive sequence to 39 skin biopsies taken from patients with leprosy of the lepromatous type and tuberculoid type. Crude DNA samples were prepared from tissue sections that were deparaffinized and subjected to proteinase-K digestion without any further treatment for DNA purification. Overcoming a false-negative reaction by an elongation of the period for enzymatic digestion and an appropriate dilution of the samples, an amplification of the target sequence was obtained as a single band with all 39 skin biopsies tested. The fragments specifically amplified by the PCR were subjected to direct sequencing and were confirmed to be identical with an internal 372 bp of M. leprae-specific repetitive sequence. Although in nine of 24 nonleprosy control samples, a false-positive amplification was observed as from one to several bands, they were distinguishable from the specific one by the electrophoretic pattern. This PCR makes up for the classic histological methods used in the diagnosis of leprosy.
Nishimura
M
M
Department of Clinical Genetics, Kyushu University, Beppu, Japan.
Kwon
K S
KS
Shibuta
K
K
Yoshikawa
Y
Y
Oh
C K
CK
Suzuki
T
T
Chung
T A
TA
Hori
Y
Y
D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005557:Formaldehyde; D006801:Humans; D007918:Leprosy / Q000175:diagnosis* / Q000382:microbiology; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology*; D016707:Tissue Fixation
D004269:DNA, Bacterial; D005557:Formaldehyde