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Indian Journal of Medical Microbiology, Vol. 28, No. 1, January-March, 2010, pp. 57-59 Brief Communication Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears RR Kamble, VS Shinde, SP Madhale, AA Kamble, BP Ravikumar, *RS Jadhav Stanley Browne Laboratory (RRK, RSJ), TLM Community Hospital, Nand Nagari Delhi - 110 093; Blue Peter Research Centre (VSS), Cheralpally, Hyderabad; Richardson Leprosy Hospital (SPM, AAK, BPR), Sangli-Miraj Road, Miraj - 416 410 Dist. Sangli. Maharashtra, India Correspondence Address: * Richardson Leprosy Hospital (SPM, AAK, BPR), Sangli-Miraj Road, Miraj - 416 410 Dist. Sangli. Maharashtra, India, rupenjadhav@yahoo.com Date of Submission: 14-Nov-2008 Code Number: mb10015 PMID: 20061767 DOI: 10.4103/0255-0857.58732 Abstract Identification of Mycobacterium leprae, which causes leprosy, is done by Ziehl Neelsen Carbol Fuchsin (ZNCF) stained slit skin smear microscopy that aids in the diagnosis and quantification of approximate bacterial load carried by the patient. We attempted M. leprae DNA extraction from 46 stained slit skin smear negative slides, using Proteinase K and SDS lysis, followed by ethanol precipitation. M. leprae specific primers (16SrRNA) were used for PCR-based amplification of DNA. We could detect M. leprae DNA in 15 (32.6%) samples. The method can be useful in the diagnosis of apparently slit skin smear negative leprosy cases. Keywords: DNA, slit skin smear, polymerase chain reaction, leprosy Introduction Leprosy, caused by Mycobacterium leprae, is a chronic granulomatous disease affecting the skin and peripheral nervous system. Nerve damage and deformity caused by the disease necessitates it to be diagnosed correctly, at the early stages, for effective treatment. Diagnosis of the disease depends on the clinical signs and symptoms and slit skin smear (SSS) positivity. SSS staining can aid in the confirmation of diagnosis in a suspect case, relapse, classification of new patients and also in monitoring the efficacy of treatment. Reproducibility of SSS results depends on individuals and expertise available, as 10 4 bacilli per gram of tissue is required for reliable detection in stained smears. [1] Polymerase Chain Reaction (PCR), being sensitive and specific, has been used for rapid and accurate detection of microorganisms in clinical samples. Detection of M. leprae in SSS by PCR has been reported [2] and shown to have advantage over both SSS staining and detection of anti-phenolic glycolipid1 antibody. [3] The PCR technology can help arrive at quick and conclusive diagnosis, identification and treatment of early cases, differentiation between leprosy and non-leprosy cases and also for epidemiological purposes. The SSS-negative mutli-bacillary patients were seen to harbor M. leprae in their tissues even after completion of 24 months of MB-MDT treatment, which makes it necessary to analyze the negative SSS carefully. [4] DNA extraction from stained slides was attempted in many infections and PCR has been successfully carried out with the extracted DNA. [5],[6] Amplification of DNA from stained SSS slides can be used to cross-check the negative slides which may, in turn, help in correct diagnosis or cross referral or even to analyze archived samples. The study attempts to extract DNA from ZNCF-stained SSS slides so as to detect the presence of M. leprae DNA by PCR in the smear negative slides. Materials and Methods Forty six archived ZNCF-stained SSS negative slides were obtained from the clinical lab. The slides collected for DNA extraction were from the last five years, stored at room temperature. Processing of slides and DNA extraction Xylene treatment was used to remove the oil from the slides. After drying, 150μl of lysis buffer (1 mg/ml Proteinase K and 0.05% Tween 20) was added on the smear and the smear was scraped using fine needle. It was then collected in a 1.5 ml microfuge tube. The tube was incubated at 60°C overnight. About 30 μl of 10% Sodium dodecyl sulfate was added to the lysate and further incubated at 60°C for one hour. The reaction was terminated at 94°C for 15 minutes. The mixture was centrifuged at 10,000 rpm for 10 minutes and the supernatant was carefully transferred to a new clean 1.5 ml microfuge tube. The supernatant was precipitated with ethanol at minus 20°C overnight The DNA was pelleted by centrifuging at 10,000 rpm for 15 minutes. The supernatant was discarded and the pellet was washed again with ethanol. The pellet was air dried and re-suspended in 30 μl of TE buffer (10 m m0 Tris, and 1mM EDTA, pH 8) and dissolved by incubating at 37°C for an hour. This DNA preparation was further used for PCR. Polymerase chain reaction for detection of M. leprae The 16S rRNA primers 7 specific for M. leprae viz. P2: CGG AAA GGT CTC TAA AAA ATC TT and P3: CAT CCT GCA CCG CAA AAA GCT T had been used to amplify 173 bp fragment of 16S rRNA gene. The PCR was performed in a total volume of 50 μl, in which 5 μl of sample, 50 ng/reaction of both the primers and 25 μl 2X PCR Premix (Bangalore Genei, Bangalore, India) was added. Reagent negative control and M. leprae DNA positive controls were used. PCR amplification conditions were same as described earlier. [7] Detection of amplified product The amplified PCR product was detected using agarose gel electrophoresis. About 10 μl of the PCR product, along with the Orange G (10 mg/ml) loading dye, was electrophoresed through 1.5% agarose gel prepared in a tris-borate EDTA buffer containing Ethidium bromide (10 mg/ml) dye at a final concentration of 0.5 μg/ml at 70 V for 2 hours. The gel was visualized using gel documentation system (Alpha Imager). Results The PCR results were interpreted by observing the presence or absence of the amplified 173-bp DNA band in an agarose gel [Figure - 1]. We observed that 15 (32.6%) samples showed amplification. Of the 46 smears, 44 were from patients classified as MB type and two were classified as PB type. Both the smears from PB patients were PCR negative [Table - 1]. The smears were collected over a span of five years and positivity by PCR did not relate to the storage period of the slide. The study observes that while Proteinase K and SDS help in effective lysis whereas ethanol precipitation and washing help in removal of the stain. Discussion Slit skin smears from leprosy patients have been used in PCR and RTPCR studies. [7],[8] Studies on the comparison between PCR-based identification and conventional SSS microscopy found PCR much more sensitive. [8],[9] In our study, this observation is confirmed as some of the SSS negative slides came out to be positive when screened by PCR. The DNA was successfully isolated from stained slides in many other diseases. [10],[11],[12] Paraffin-embedded tissues, Papanicolaou or Giemsa-stained smears have been proved to be suitable for DNA isolation. [10] DNA extraction from ZNCF-stained slides in tuberculosis (TB) has been done to retrospectively study drug resistance and facilitate specimen transportation. [11] This study shows that DNA extraction for PCR can be done from ZNCF- stained SSS slides. The PCR results with SSS negative samples show that this method can be used to retrospectively analyze the archived slides. The DNA extraction and PCR from ZNCF-stained skin smear slides can be of added advantage in the screening of smear negative slides in leprosy, which will help in administering adequate treatment. Acknowledgment We wish to acknowledge the support of The Leprosy Mission, India, to carry out this work. We also acknowledge Dr.Jeyakumar Daniel, Director, TLM India, Dr.P.S.S.Sundar Rao, Research Coordinator, TLM, India for guidance and Mrs. Mallika Lavania for help in the preparation of this manuscript. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10015f1.jpg] [mb10015t1.jpg] |
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