Polymerase Chain Reaction-restriction Enzyme Analysis Research Articles

Identification of Nontuberculous Mycobacterium Species by Polymerase Chain Reaction - Restriction Enzyme Analysis (PCR-REA) of rpoB gene in Clinical Isolates.

Nontuberculous mycobacteria (NTM) infections are an emerging global health concern with increasing incidence. Conventional identification methods for NTM species in clinical settings are prone to errors. This study evaluates a newer method, polymerase chain reaction-restriction enzyme analysis (PCR-REA) of the rpoB gene, for NTM species identification. The study identified NTM species in clinical samples using conventional biochemical techniques and compared the results with PCR-REA of the rpoB gene. This cross-sectional study was conducted at a tertiary health-care center in North India over 18 months, analyzing both pulmonary and extrapulmonary samples. Two hundred and forty-seven NTM isolates were identified using phenotypic and biochemical methods. The same isolates were subjected to rpoB gene amplification by PCR followed by REA using Msp I and Hae III enzymes. Conventional methods identified 12 different NTM species (153 slow-growing and 94 rapid-growing), whereas PCR-REA identified 16 species (140 slow-growing, 107 rapid-growing). The Mycobacterium avium intracellulare complex was the most common species isolated. PCR-REA demonstrated higher resolution in species identification, particularly in differentiating within species complexes. PCR-REA of the rpoB gene proves to be a simple, rapid, and more discriminative tool for NTM species identification compared to conventional methods. This technique could significantly improve the diagnosis and management of emerging NTM infections in clinical settings.

Molecular Identification, and Characterization of Mycobacterium kansasii Strains Isolated from Four Tuberculosis Regional Reference Laboratories in Iran During 2016-2018.

BackgroundNon-tuberculous mycobacterial (NTM) infections are growing concern in many countries around the globe including Iran. Among them, Mycobacterium kansasii (M. kansasii) causes both pulmonary and extra-pulmonary infections. Despite the high prevalence of M. kansasii isolates in Iran, unfortunately little is known about the epidemiological aspects of M. kansasii infection. Hence, the aim of the present study was to investigate the molecular identification, determination of subtypes variation and geographic distribution of clinical isolates of M. kansasii isolates.MethodsIn the present study, 108 clinical pulmonary isolates suspected to NTM were collected from four Tuberculosis Regional Reference Laboratories in Iran during 2016–2018. The isolates were confirmed as NTM using conventional and molecular methods. Among them, M. kansasii isolates were subjected to rpoB gene sequencing. For determination of subtyping of M. kansasii isolates, polymerase chain reaction-restriction enzyme analysis (PCR-REA) based on the hsp65 gene was performed.ResultsBased on the rpoB gene sequence analysis, 33 (30.5%) isolates were identified as M. kansasii species, compared to 31 (28.7%) isolates using phenotypic methods. The subtype I was the most frequent subtype (n=24; 72.7%), followed by subtype II (n=8; 24.2%).ConclusionWe indicated that the rate of M. kansasii isolation with clinical significance appears to be increasing in Iran, especially in highly industrialized cities. The high rate of M. kansasii subtype I may suggest that this genotype has a particular potency for colonization, and a higher epidemiological potential for causing infection in humans. More studies are needed to provide a better understanding of the biology and pathogenicity of M. kansasii subtype I.

Identification of the Infection Source of an Outbreak of Mycobacterium Chelonae Keratitis After Laser in Situ Keratomileusis

Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes. In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections. Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes. Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

Pseudooutbreak of rapidly growing mycobacteria due to Mycobacterium abscessus subsp bolletii in a digestive and respiratory endoscopy unit caused by the same clone as that of a countrywide outbreak.

The nontuberculous mycobacteria (NTM) are widely spread. In Brazil, 2,520 cases of rapidly growing mycobacteria (RGM) infections after medical procedures were reported, with 5.4% of cases related to nonsurgical invasive procedures and with an occurrence of 1 clone (BRA100) of Mycobacterium abscessus subsp bolletii. To describe a pseudooutbreak of M abscessus subsp bolletii in an endoscopy and bronchoscopy unit. The alert for a pseudooutbreak was given when 3 patients, in the same week, had a positive bronchoalveolar lavage culture for M abscessus subsp bolletii. The patients had no symptoms/signs of mycobacterial infection; thus, contamination of bronchoscopes was suspected. Samples for culturing were collected from bronchoscopes, digestive endoscopes, automated disinfection machines, and the water supply. Clinical samples were identified by polymerase chain reaction restriction-enzyme analysis (PRA) of the hsp65 gene and their pulsed-field gel electrophoresis pattern was compared with environmental samples. The investigation demonstrated a contamination of bronchoscopes, digestive endoscopes, and disinfection machines. Molecular typing demonstrated that all strains belonged to the same clone (MAB01), identical to clone BRA100. Cross-transmission due to poor disinfection as well as resistance to glutaraldehyde may play roles in the spread of MAB01 M abscessus subsp bolletii, which may have a unique resistance to the environment and adaption to human hosts. However the water supply may have played a role. Attention is needed to ensure the quality of water used to rinse disinfected equipment.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Genotyping of Mycobacterium tuberculosis complex based on restriction enzyme analysis of the hsp65 gene

Several genotypes of Mycobacterium with varied degree of virulence and drug resistance exist globally. Grouping of the genotypes into principal genetic groups is critical for tuberculosis (TB) control. This study determined the suitability of polymerase chain reaction- restriction enzyme analysis (PCR-REA) technique as an epidemiological typing tool and a technique for drug resistance surveillance of TB. Seventy-five Mycobacterium tuberculosis isolates from National TB Reference Laboratory, Nigeria Institute of Medical Research (NIMR) Yaba, Lagos, Nigeria were identified and their susceptibility to isoniazid and rifampicin determined using Genotype MTBDRplus technique. Isolates were further confirmed by polymerase chain reaction (PCR) with primer specific to 65 kDa heat shock protein (hsp65) of Mycobacterium species, Fingerprinting was done by restriction enzyme (Bst Ell) analysis. Of the 75 isolates studied, 5(6.7%) were multi-drug resistant (MDR)- TB, 11(14.7%) and 7(9.3%) were mono-resistant to rifampicin and isoniazid respectively. PCR-REA profile revealed 3 different patterns. Majority (96.2%) of the susceptible strains and 100% each of mono-resistant and MDR -TB yielded 2 fragments of 250 and 120 bp. Our findings demonstrated that hsp65 kDa based molecular genotyping technique is a rapid, simple and easy-to-interpret method and may be used as a predictor of M. tuberculosis genetic variation. Key words: Genotyping, Mycobacterium tuberculosis, heat shock protein, restriction enzyme analysis, drug susceptibility.

Occurrence of pathogenic environmental mycobacteria on surfaces in health institutions

Occurrence of environmental mycobacteria pathogenic on surfaces in health institutions Maria Gorete Mendes dé souza1, Daisy Nakamura Sato2, Clarice Queico Fujimura Leite3, Sérgio Roberto de Andrade Leite4, Flávio Garcia Sartori1, Karina de Andrade Prince3, Luciana Assirati Casmeiro1, Carlos Henrique Gomes Martins11Laboratório de Pesquisa em Microbiologia Aplicada, Universidade de Franca, Franca, Brazil; 2Laboratório de Micobactéria, Instituto Adolfo Lutz, Ribeirão Preto, Brazil; 3Departamento de Ciências Biológicas, Universidade Estadual Paulista, Araraquara, Brazil; 4Instituto de Química, Universidade Estadual Paulista, Araraquara, BrazilAbstract: The presence of environmental mycobacteria on surfaces in two public health institutions, namely a health center and a hospital in upstate São Paulo (Brazil), was identified by polymerase chain reaction-restriction enzyme analysis (PRA). The possible sources of contamination by these microorganisms were evaluated, contributing to epidemiology studies.Methods: From June 2005 to June 2006, a total of 632 samples were collected from exposed surfaces, such as washbasins, drinking fountains, and other accessible sites, and the mycobacteria present in the samples were isolated and cultured.Results: Sixty-five mycobacteria were isolated from the 632 samples; 47 of which were detected in samples from the health center and 18 in samples collected from the hospital. The isolates were identified by DNA restriction patterns obtained by PRA, and potentially pathogenic species were found to be prevalent among the identified mycobacteria. This study shows that the PRA technique can be employed as a fast and easy method for identification of nontuberculous mycobacteria in public areas.Conclusions: The isolation of environmental mycobacteria from the two health institutions demonstrates that these surfaces are reservoirs of potentially pathogenic mycobacteria and indicates the need for continuous maintenance and monitoring. These data will add to the study of the epidemiology of these microorganisms.Keywords: environmental mycobacteria, health center, PCR, PRA

Isolamento de micobactérias não-tuberculosas em São José do Rio Preto entre 1996 e 2005

To study the incidence of nontuberculous mycobacteria and the range of species isolated between 1996 and 2005 at a regional branch of the Adolfo Lutz Institute-located in the city of São José do Rio Preto, Brazil-and to show the importance of laboratory testing. Mycobacteria were isolated from pulmonary and extrapulmonary specimens and identified through phenotyping and molecular methods (polymerase chain reaction-restriction enzyme analysis). We isolated 317 nontuberculous mycobacterium strains: Mycobacterium avium complex, 182 (57.4%); M. gordonae, 33 (10.4%); M. fortuitum, 25 (7.9%); M. chelonae, 8 (2.5%); M. terrae complex, 8(2.5%); M.kansasii, 7 (2.2%); and less frequent species, 54 (17%). During this period, 72 cases (33.3%) were characterized as mycobacteriosis, according to bacteriological criteria established by the American Thoracic Society in 2007. Of those 72 cases, 56 were attributed to M.avium complex. Of those 56, 29 (51.8%) were characterized as disseminated disease. Six cases were attributed to M. fortuitum, 3 to M. gordonae, 2 to M. chelonae, 1 to M. abscessus, 1 to M. kansasii, 1 to M. intracellulare, 1 to M. malmoense and 1 to Mycobacterium ssp. These results show the importance of the bacteriological diagnosis, since identification of the species enables early and appropriate treatment.

Comparative evaluation of Polymerase Chain Reaction–Restriction Enzyme Analysis (PRA) and sequencing of heat shock protein 65 ( hsp65) gene for identification of aquatic mycobacteria

Traditional identification of mycobacteria based on cultural and biochemical tests can take several weeks and may fail to provide a precise identification. Polymerase Chain Reaction–Restriction Enzyme Analysis (PRA) of the gene encoding heat shock protein 65 kDa ( hsp65) gene has been proposed as a rapid and inexpensive alternative approach. Despite being widely used for differentiation of mammalian mycobacteria, this method has only been applied in the identification of a small number of aquatic mycobacteria. The present study aimed to evaluate the potential use of PRA of hsp65 for the identification of aquatic mycobacteria compared with sequence analysis. Seventy one mycobacterial isolates including, 10 type/reference strains and the remainder field isolates, were subjected to PRA of a 441 bp fragment of this gene. For 68 representative isolates, sequence analysis was performed. All rapidly and slowly growing mycobacteria had best matches with 99.3% to 100% similarity with their corresponding species in the databanks. PRA proved to be a simple and rapid method for identifying aquatic mycobacteria. However, the incidence of similar or identical restriction patterns for some species of mycobacteria, and in particular, identification of new species of mycobacteria is a major problem using such a method. In contrast, the nucleic acid sequencing of the hsp65 gene yielded unambiguous results.

Carriage ofMalasseziaspp. yeasts in cats with diabetes mellitus, hyperthyroidism and neoplasia

The frequencies of isolation and population sizes of Malassezia spp. on skin and at mucosal sites in 16 cats with diabetes mellitus, 20 cats with hyperthyroidism and 8 cats with neoplasia did not vary significantly from those of healthy cats when measured with the use of contact plates and a swab technique. M. pachydermatis was isolated from nine sites in one cat with feline paraneoplastic alopecia and pancreatic adenocarcinoma, two cats with diabetes mellitus and five cats with hyperthyroidism. A polymerase chain reaction-restriction enzyme analysis (PCR-REA) method that differentiated the 11 species of Malassezia spp was used to identify the lipid-dependent isolates that were obtained from two cats with diabetes mellitus, two cats with hyperthyroidism and one cat with multicentric lymphoma. Six isolates had PCR-REA patterns that were indistinguishable from M. slooffiae CBS 7956 and three matched M. nana CBS 9557. Our data suggests that skin and mucosal counts of Malassezia spp. are not routinely increased in cats with diabetes mellitus or hyperthyroidism but we report a further example of an association between feline paraneoplastic alopecia and Malassezia spp. proliferation. To the authors' knowledge, this is the first report of the isolation of M. slooffiae from feline skin.

Study of the ITS region in an atypical isolate and comparison with six species of Microsporum

Microsporum species are a frequent cause of cutaneous mycoses in humans. Atypical strains of Microsporum can sometimes be difficult to identify with conventional methods. Recently, we have obtained a Microsporum isolate with atypical morphology and special nutritional requirements (Microsporum CHUS-126-02). As several molecular techniques have been developed for the identification of fungi, we analysed six Microsporum species (M. canis, M. gypseum, M. gallinae, M. nanum, M. ferrugineum and M. persicolor) in order to compare them with our isolate, by using polymerase chain reaction-restriction enzyme analysis (PCR-REA). We studied the nucleotide sequence of the internal transcribed spacer regions from the nuclear DNA encoding for the ribosomal domain. Digestion with MvaI and EcoRI endonucleases obtained specific patterns for M. gypseum, M. gallinae, M. nanum and M. persicolor. Microsporum canis, M. ferrugineum and Microsporum CHUS-126-02 yielded the same patterns. Based on these results and phenotypic criteria, we classified our atypical isolate as M. canis.

Phenotypical and molecular characterization of Microsporum canis strains in north-east Brazil

This study investigated the possible correlation between the phenotypical and genotypical characteristics of Microsporum canis isolated from cats and dogs in north-east Brazil. The mycological study was conducted by direct microscopic examination and by fungal culture. Polymerase chain reaction-restriction enzyme analysis and random amplification of polymorphic DNA techniques were used for the genotypical analysis. The morphological analysis showed a considerable diversity of colonies as well as different morphologies of conidia, despite the M. canis strains having been isolated under the same conditions. However, the molecular analysis showed that all analysed strains are genetically similar. This study, based on phenotypical and molecular analysis, evidences the wide spectrum of phenotypical variations in M. canis in contrast to the stable genotypes of such dermatophytes. The findings of this study indicate that M. canis isolated from cats and dogs with dermatophytosis in north-east Brazil may be clones, well adapted to the conditions of this region, despite M. canis showing different morphological features.

Genetic diversity of Mycobacterium avium subspecies paratuberculosis isolates from goats detected by pulsed-field gel electrophoresis

Paratuberculosis in goats occurs worldwide causing considerable economic losses mainly due to reduced milk production. Nowadays, there is still relatively little knowledge about the epidemiology of this disease in goats, and only a few epidemiological studies have been carried out in goats naturally infected with Mycobacterium avium subspecies paratuberculosis ( M. a. paratuberculosis). The objective of this study was to characterize forty four clinical caprine isolates of M. a. paratuberculosis by different molecular techniques (pulsed-field gel electrophoresis [PFGE], restriction fragment length polymorphism analysis coupled with hybridization to IS 900, and IS 1311 polymerase chain reaction-restriction enzyme analysis) to determine the most useful technique for molecular typing of caprine isolates, as well as to disclose the genetic variation amongst caprine isolates and the relationship with strains isolated from other animal species. PFGE was found to be the most discriminative technique identifying a total of 13 ‘multiplex’ PFGE profiles, ten of which were novel profiles found only in caprine isolates to date. All isolates were genotyped as Type II strains, except two isolates that resembled the intermediate group referred as Type III.

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.

Species identification by genotyping and determination of antibiotic resistance in Campylobacter jejuni and Campylobacter coli from humans and chickens in Sweden

Campylobacter is today the most common cause of human bacterial enteritis in Sweden, as well as in most other industrialized countries. Common sources of infection are undercooked chicken meat, unpasteurized milk and contaminated drinking water. One aim with our present study was to identify the species Campylobacter jejuni and Campylobacter coli strains from humans and chickens using a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method, as well as traditional hippurate hydrolysis test. Another aim was to investigate the antibiotic resistance pattern of the human domestic C. jejuni/ C. coli isolates from infected patients and isolates from healthy Swedish chicken, as well as isolates from humans infected abroad. If discrimination between C. jejuni and C. coli was based on testing for hippurate hydrolysis, 95% of the human domestic strains and 88% of the chicken strains were identified as C. jejuni. Based on genotyping by PCR/REA, 100% of the human domestic strains and 98% of the chicken strains were attributed to C. jejuni. The E-test and disc diffusion methods were used for phenotypic antibiotic resistance studies. The two methods gave similar results. Most Swedish C. jejuni/ C. coli isolates both from humans and chickens were sensitive to doxycycline and erythromycin, which are antibiotics used to treat human infection. Only 7% of the human domestic strains and 2% of the chicken strains were resistant to the quinolones tested. As a comparison, more than 94% of strains isolated from travelers to Asia and southern Europe showed antibiotic resistance to one or more drugs.

An outbreak of Mycobacterium chelonae infection following liposuction.

Among 82 patients who underwent liposuction performed by a single practitioner in a 6-month period, 34 (41%) developed cutaneous abscesses. An organism identified as Mycobacterium chelonae by polymerase chain reaction restriction-enzyme analysis was recovered from cultures of samples from 12 of those patients. DNA large restriction-fragment pattern analysis by pulsed-field gel electrophoresis demonstrated that a strain of M. chelonae recovered from biofilm in the piped-water system in one of the physician's offices differed by only 2 restriction fragments from the 12 patient isolates, which differed from each other by 0 or 1 restriction fragment. A detailed retrospective cohort study that included interviews with former employees and statistical analysis of risk factors indicated that inadequate sterilization and rinsing of surgical equipment with tap water were likely sources of mycobacterial contamination. This is the first reported outbreak of nosocomial infection due to M. chelonae in which a source has been identified and the first to occur in association with liposuction in patients in the United States.

Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis

Onychomycosis, a fungal nail infection, has become one of the most important dermatophytoses. Unfortunately, a predictably successful diagnostic approach to onychomycosis does not yet exist. The purpose of this study was to develop a deoxyribonucleic acid (DNA)-based diagnostic method to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis. We attempted to detect fungi in the nail using polymerase chain reaction (PCR) primer systems that were designed in conserved sequences of the small ribosomal subunit 18S-rRNA genes shared by most fungi, and differentiated between species by restriction enzyme analysis of the amplified product. Fragments of the gene coding for 18S-rRNA were amplified successfully from medically important fungi species, but not from normal nails. Restriction fragment length polymorphism patterns using HaeIII endonuclease were sufficiently different to allow the recognition of individual species. The PCR-restriction enzyme analysis method appears to be a more sensitive detection and identification technique for onychomycosis than conventional methods, and has considerable diagnostic value.

Differentiation of Australian cucumber mosaic cucumovirus isolates: comparison of dsRNA type, polymerase chain reaction-restriction enzyme analysis, DNA hybridisation and serogrouping

We examined the differentiation of 22 Australian isolates of cucumber mosaic cucumovirus (CMV) by comparing PAGE mobilities of the three RNAs (dsRNA mobilities), serogroups using monoclonal antibodies, dot blot hybridisation (DBH) with subgroup-specific RNA 3 probes and PCR-restriction enzyme analysis (Msp I) of the coat protein gene. While there was a high degree of correlation between the results obtained with all four methods, we found exceptions with 2 of the 22 isolates analysed. This indicates that more than one test is necessary to obtain reliable information about the identity of a CMV isolate.

Diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes in a Chinese family by PCR/restriction enzyme analysis

The clinical presentation and the biochemical and molecular genetic findings are described in a 13 year old Chinese boy with MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). The diagnosis was initially suspected because of the characteristic clinical features and the strong family history of convulsions. Using polymerase chain reaction-restriction enzyme analysis, the heteroplasmic nt3243 A-->G mutation in mtDNA of peripheral blood leucocytes and a muscle sample was demonstrated. The oligosymptomatic relatives were then screened by this method and the degree of heteroplasmy was analysed. This appears to be the first report of a MELAS family in Hong Kong with this described mutation. Molecular genetic techniques are advantageous in the diagnosis of MELAS.

Molecular analysis of T cell receptor Vβ chain to detect leukemia cell clonality in patients by adaptor ligation-mediated polymerase chain reaction

We have developed a simple and rapid method to analyze the clonality of leukemia cells. After three rounds of amplification by adaptor-ligation polymerase chain reaction (PCR), the cDNA is cut with AluI, HaeIII, RsaI, and Sau3AI, and analyzed by polyacrylamide gel electrophoresis. The size of the restriction fragments is compared to that of the published restriction fragments size each TCR-β subfamily V region. The sensitivity of adaptor-ligation PCR restriction enzyme analysis (AL-PCR-REA) was 10 −4 MOLT-4 T-ALL cell population in the normal peripheral blood lymphocytes (PBL). Application of AL-PCR-REA to PBL and bone marrow (BM) cells from eight clinical leukemia samples indicated that a detection sensitivity was rather low, but revealed the clonality of all eight clinical samples. This AL-PCR-REA method can detect clonality without the need for either radioisotopes or sequencing procedures.