Delineation of loci governing an extra-earliness trait in lentil (Lens culinaris Medik.) using the QTL-Seq approach.

Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole-genome resequencing (WGRS) based QTL-seq identified the loci governing earliness in lentil. The genetic analysis for maturity duration provided a good fit to 3:1 segregation (F2), indicating earliness as a recessive trait. WGRS of Globe Mutant (late parent), late-flowering, and early-flowering bulks (from RILs) has generated 1124.57, 1052.24 million raw and clean reads, respectively. The QTL-Seq identified three QTLs (LcqDTF3.1, LcqDTF3.2, and LcqDTF3.3) on chromosome 3 having 246244 SNPs and 15577 insertions/deletions (InDels) and 13 flowering pathway genes. Of these, 11 exhibited sequence variations between bulks and validation (qPCR) revealed a significant difference in the expression of nine candidate genes (LcGA20oxG, LcFRI, LcLFY, LcSPL13a, Lcu.2RBY.3g060720, Lcu.2RBY.3g062540, Lcu.2RBY.3g062760, LcELF3a, and LcEMF1). Interestingly, the LcELF3a gene showed significantly higher expression in late-flowering genotype and exhibited substantial involvement in promoting lateness. Subsequently, an InDel marker (I-SP-383.9; LcELF3a gene) developed from LcqDTF3.2 QTL region showed 82.35% PVE (phenotypic variation explained) for earliness. The cloning, sequencing, and comparative analysis of the LcELF3a gene from both parents revealed 23 SNPs and InDels. Interestingly, a 52 bp deletion was recorded in the LcELF3a gene of L4775, predicted to cause premature termination of protein synthesis after 4 missense amino acids beyond the 351st amino acid due to the frameshift during translation. The identified InDel marker holds significant potential for breeding early maturing lentil varieties.

Factors causing stress among students of a medical college in Kolkata, India.

BACKGROUND: Medical students feel a significant amount of stress due to a variety of factors. Few studies have explored the relative size of these various stressors to identify which are most important. This study was undertaken to quantify the magnitude of various sources of stress among students of a medical college at Kolkata, West Bengal, and also to assess the reliability of Medical Students' Stressor Questionnaire (MSSQ-40) in this context. METHODS: We evaluated the degree of stress along various dimensions using the MSSQ-40, a scale to measure stress among medical students that has been validated in other countries. Differences in stress and its causal factors were analyzed across demographic subgroups. The reliability of the MSSQ-40 was evaluated using Cronbach's alpha. RESULTS: The overall prevalence of stress was 91.1% and the vast majority of students (94.9%) were stressed due to academic reasons. Academic related stress was found to be higher among students who are not conversant with the local language, experienced a change in medium of teaching from secondary school to medical school, and resided in a hostel. The MSSQ-40 in general, and its academic-related stress domain specifically, were found to be reliable in our setting. DISCUSSION: Early detection and remedying stressors will help to build physical and mental health in medical students. Language training early during the medical course might reduce academic stress among our students. Further studies should relate individuals' stress with their academic performance.

Pyrazole-oxadiazole conjugates: synthesis, antiproliferative activity and inhibition of tubulin polymerization.

A number of pyrazole-oxadiazole conjugates were synthesized and evaluated for their ability to function as antiproliferative agents on various human cancer cell lines. These conjugates are comprised of pyrazole and oxadiazole scaffolds closely attached to each other without any spacer as two structural classes. The Type I class has a trimethoxy substituent and the type II class has a 3,4-(methylenedioxy) substituent on their A rings. Among these conjugates 11a, 11d and 11f manifest potent cytotoxicity with IC50 values ranging from 1.5 µM to 11.2 µM and inhibit tubulin polymerization with IC50 values of 1.3 µM, 3.9 µM and 2.4 µM respectively. The cell cycle assay showed that treatment with these conjugates results in accumulation of cells in the G2/M phase and disrupts the microtubule network. Elucidation of zebrafish embryos revealed that the conjugates cause developmental defects. Molecular docking simulations determined the binding modes of these potent conjugates at the colchicine site of tubulin.

Identification of quantitative trait loci (QTLs) regulating leaf SPAD value and trichome density in mungbean (Vigna radiata L.) using genotyping-by-sequencing (GBS) approach.

Quantitative trait loci (QTL) mapping is used for the precise localization of genomic regions regulating various traits in plants. Two major QTLs regulating Soil Plant Analysis Development (SPAD) value (qSPAD-7-1) and trichome density (qTric-7-2) in mungbean were identified using recombinant inbred line (RIL) populations (PMR-1×Pusa Baisakhi) on chromosome 7. Functional analysis of QTL region identified 35 candidate genes for SPAD value (16 No) and trichome (19 No) traits. The candidate genes regulating trichome density on the dorsal leaf surface of the mungbean include VRADI07G24840, VRADI07G17780, and VRADI07G15650, which encodes for ZFP6, TFs bHLH DNA-binding superfamily protein, and MYB102, respectively. Also, candidate genes having vital roles in chlorophyll biosynthesis are VRADIO7G29860, VRADIO7G29450, and VRADIO7G28520, which encodes for s-adenosyl-L-methionine, FTSHI1 protein, and CRS2-associated factor, respectively. The findings unfolded the opportunity for the development of customized genotypes having high SPAD value and high trichome density having a possible role in yield and mungbean yellow vein mosaic India virus (MYMIV) resistance in mungbean.

Genome-wide discovery of InDels and validation of PCR-Based InDel markers for earliness in a RIL population and genotypes of lentil (Lens culinaris Medik.).

The systematic identification of insertion/deletion (InDel) length polymorphisms from the entire lentil genome can be used to map the quantitative trait loci (QTL) and also for the marker-assisted selection (MAS) for various linked traits. The InDels were identified by comparing the whole-genome resequencing (WGRS) data of two extreme bulks (early- and late-flowering bulk) and a parental genotype (Globe Mutant) of lentil. The bulks were made by pooling 20 extreme recombinant inbred lines (RILs) each, derived by crossing Globe Mutant (late flowering parent) with L4775 (early flowering parent). Finally, 734,716 novel InDels were identified, which is nearly one InDel per 5,096 bp of lentil genome. Furthermore, 74.94% of InDels were within the intergenic region and 99.45% displayed modifier effects. Of these, 15,732 had insertions or deletions of 20 bp or more, making them amenable to the development of PCR-based markers. An InDel marker I-SP-356.6 (chr. 3; position 356,687,623; positioned 174.5 Kb from the LcFRI gene) was identified as having a phenotypic variance explained (PVE) value of 47.7% for earliness when validated in a RIL population. Thus, I-SP-356.6 marker can be deployed in MAS to facilitate the transfer of the earliness trait to other elite late-maturing cultivars. Two InDel markers viz., I-SP-356.6 and I-SP-383.9 (chr. 3; linked to LcELF3a gene) when tested in 9 lentil genotypes differing for maturity duration, clearly distinguished three early (L4775, ILL7663, Precoz) and four late genotypes (Globe Mutant, MFX, L4602, L830). However, these InDels could not be validated in two genotypes (L4717, L4727), suggesting either absence of polymorphism and/or presence of other loci causing earliness. The identified InDel markers can act as valuable tools for MAS for the development of early maturing lentil varieties.

Genome-wide association studies for phenological and agronomic traits in mungbean (Vigna radiata L. Wilczek).

Mungbean (Vigna radiata L. Wilczek) is one of the important warm-season food legumes, contributing substantially to nutritional security and environmental sustainability. The genetic complexity of yield-associated agronomic traits in mungbean is not well understood. To dissect the genetic basis of phenological and agronomic traits, we evaluated 153 diverse mungbean genotypes for two phenological (days to heading and days to maturity) and eight agronomic traits (leaf nitrogen status using SPAD, plant height, number of primary branches, pod length, number of pods per plant, seeds per pod, 100-seed weight, and yield per plant) under two environmental conditions. A wide array of phenotypic variability was apparent among the studied genotypes for all the studied traits. The broad sense of heritability of traits ranged from 0.31 to 0.95 and 0.21 to 0.94 at the Delhi and Ludhiana locations, respectively. A total of 55,634 genome-wide single nucleotide polymorphisms (SNPs) were obtained by the genotyping-by-sequencing method, of which 15,926 SNPs were retained for genome-wide association studies (GWAS). GWAS with Bayesian information and linkage-disequilibrium iteratively nested keyway (BLINK) model identified 50 SNPs significantly associated with phenological and agronomic traits. In total, 12 SNPs were found to be significantly associated with phenological traits across environments, explaining 7%-18.5% of phenotypic variability, and 38 SNPs were significantly associated with agronomic traits, explaining 4.7%-27.6% of the phenotypic variability. The maximum number of SNPs (15) were located on chromosome 1, followed by seven SNPs each on chromosomes 2 and 8. The BLAST search identified 19 putative candidate genes that were involved in light signaling, nitrogen responses, phosphorus (P) transport and remobilization, photosynthesis, respiration, metabolic pathways, and regulating growth and development. Digital expression analysis of 19 genes revealed significantly higher expression of 12 genes, viz. VRADI01G08170, VRADI11G09170, VRADI02G00450, VRADI01G00700, VRADI07G14240, VRADI03G06030, VRADI02G14230, VRADI08G01540, VRADI09G02590, VRADI08G00110, VRADI02G14240, and VRADI02G00430 in the roots, cotyledons, seeds, leaves, shoot apical meristems, and flowers. The identified SNPs and putative candidate genes provide valuable genetic information for fostering genomic studies and marker-assisted breeding programs that improve yield and agronomic traits in mungbean.

Genetic Gain in Yield and Associated Changes in Agronomic Traits in Wheat Cultivars Developed Between 1900 and 2016 for Irrigated Ecosystems of Northwestern Plain Zone of India.

Knowledge about the yield gain over the years due to associated changes in the yield component traits is essential for a critical understanding of yield-limiting factors. To estimate genetic gain in grain yield (GY) and component agronomic traits of wheat varieties released between 1900 and 2016 for northwestern plain zone (NWPZ) of India and to identify agronomic and/or genetic basis of the realized gains, two sets of wheat varieties comprising mega varieties and two recently developed varieties were evaluated under timely sown, tilled, and early sown conservation agriculture (CA) conditions for four consecutive years under irrigated conditions. The average annual genetic gain in GY since 1,905 under timely sown irrigated conditions was found to be 0.544% yr-1 over the average of all varieties and 0.822% yr-1 (24.27 kg ha-1 yr-1) over the first released variety, NP4. The realized mean yield increased from 2,950 kg ha-1 of the variety NP4 released in 1,905-5,649 kg ha-1 of HD3086 released in 2014. Regression analysis revealed a linear reduction in height and peduncle length (PL) over the years with a simultaneous and linear increase in biomass at the rate of 43.9 kg ha-1 yr-1 or relatively at 0.368% yr-1 mainly because of delayed heading and increased crop duration. Regression analysis showed no linear trend for tiller number and thousand-grain weight (TGW). Though harvest index (HI) was found to linearly increase relatively at the rate of 0.198% per annum, polynomial regression improved the fitness of data with the indication of no increase in HI since 1982. Interestingly, genetic gain evaluation under early sown CA conditions for 4 years showed similar relative gain (RG) [a relative improvement in varieties across breeding periods (BP)] (0.544% yr-1) but with a higher absolute value (29.28 kg ha-1 yr-1). Major mega varieties like Kalyan Sona, HD2009, PBW 343, HD2967, and HD3086, which occupied a comparatively larger area, were found highly plastic to the improvements in the production environment under timely sown conditions.

Multiplex PCR technique could be an alternative approach for early detection of leprosy among close contacts--a pilot study from India.

BACKGROUND: Implementation of Multi drug Therapy (MDT) regimen has resulted in the decline of the total number of leprosy cases in the world. Though the prevalence rate has been declining, the incidence rate remains more or less constant and high in South East Asian countries particularly in India, Nepal, Bangladesh, Pakistan and Srilanka. Leprosy, particularly that of multibacillary type spreads silently before it is clinically detected. An early detection and treatment would help to prevent transmission in the community. Multiplex PCR (M-PCR) technique appears to be promising towards early detection among contacts of leprosy cases. METHODS: A total of 234 paucibacillary (PB) and 205 multibacillary (MB) leprosy cases were studied in a community of an endemic area of Bankura district of West Bengal (Eastern India). They were assessed by smear examination for acid-fast bacilli (AFB) and M-PCR technique. These patients were treated with Multidrug Therapy (MDT) as prescribed by WHO following detection. A total of 110 MB and 72 PB contacts were studied by performing M-PCR in their nasal swab samples. RESULTS: 83.4% of MB patients were observed to be positive by smear examination for AFB and 89.2% by M-PCR. While 22.2% of PB patients were found to be positive by smear examination for AFB, 80.3% of these patients were positive by M-PCR. Among leprosy contacts (using M-PCR), 10.9% were found to be positive among MB contacts and 1.3% among PB contacts. Interestingly, two contacts of M-PCR positive MB cases developed leprosy during the period of two years follow up. CONCLUSION: The M-PCR technique appears to be an efficient tool for early detection of leprosy cases in community based contact tracing amongst close associates of PB and MB cases. Early contact tracing using a molecular biology tool can be of great help in curbing the incidence of leprosy further.

Evaluation of Endocrine Parameters as Predictor of Major Depressive Disorder.

BACKGROUND: The diagnosis of the disease, major depressive disorder (MDD), entirely depends on the presence of some symptoms without any biochemical parameter to support it. Depletion of dopamine though is an established feature, is not the sole causative factor of MDD. Moreover, it has very little diagnostic value due to a short half-life. Other chemical messengers like hormones have also been found to get altered due to significant over activity of hypothalamo-pituitary axis. Literature review suggests that cortisol, thyroid-stimulating hormone (TSH), and prolactin (PRL) are mostly altered in MDD, which can be utilized to diagnose the condition. MATERIALS AND METHODS: A total of 101 patients suffering from MDD along with 106 age- and sex-matched controls were included in this study. Cortisol, TSH, and PRL were assayed in all the study participants by enzyme immunoassay. Student's t-test and linear discriminant analysis were used for statistical analysis. RESULTS: All the three hormones were found to be significantly high in cases with MDD. When applied for classification purpose, the errors in training group were found to be 15% and 15.74% from test set. None of the normal population was wrongly diagnosed as a patient of depression. CONCLUSION: To the best of our knowledge, this is the first attempt to evaluate multiple biochemical parameters as diagnostic marker of MDD. The study is in progress to find out a cutoff value of the responsible parameter so that they can be optimally used to diagnose a case of MDD.

Role of oxidative stress and antioxidants in aetiopathogenesis and management of oral submucous fibrosis.

Lipid peroxidation product, malonaldehyde (MDA) and antioxidants were estimated in plasma and erythrocytes of 34 cases of oral submucous fibrosis (OSMF) of different grades with equal number of healthy controls to evaluate the association of reactive oxygen species (ROS) and OSMF. While plasma MDA was found to be significantly higher in patients (3.3±0.4 nmole/ml, P<0.001) as compared to controls (2.4±0.5 nmole/ml), plasma beta carotene and vitamin E levels were found to be decreased significantly in patients (81.7±14.3 µg/100 ml, P<0.001; 9.3±0.9 mg/L, P<0.01 respectively) with respect to healthy controls (110±20.8 µg/100 ml and 10.1±1.2 mg/L). The decrease in beta-carotene and vitamin E was found to be more significant in OSMF grade II and III than in grade I. After 6 weeks of oral administration of beta-carotene and vitamin E, patients showed increase in plasma level of these two antioxidants along with decrease in MDA level associated with clinical improvement.

Depression and type D personality among undergraduate medical students.

CONTEXT: Academic pressure, though established, is an unavoidable cause of depression in medical students. Role of Type D personality as determinant of depression is a new approach to the problem. AIM: Determination of relationship between Type D personality and Depression among medical students. SETTING AND DESIGN: Undergraduate students (both male and female, total 150) of Midnapore Medical College. MATERIALS AND METHODS: Beck Depression Inventory for depression and DS 14 for type D personality. STATISTICAL ANALYSIS: The scores were expressed as mean + SD. The significance of difference between the scores was done by Fisher's 2 sample t test. RESULTS AND CONCLUSION: Prevalence rate of depression was 45.3%, which was mostly of mild type (34%). Type D personality was present in 70% cases of which 15.3% had only negative affectivity, 23.3% had only social inhibition and 31% had both the components. Both depression & Type D personality were present in 36% cases. Negative affectivity component was significantly associated with depression which could be therapeutically controlled.

Synthesis, biological evaluation of new oxazolidino-sulfonamides as potential antimicrobial agents.

A number of linezolid-like oxazolidino-sulfonamides (7a-y and 8a-b) were designed and synthesized with a view to develop antimicrobial agents with improved properties. Most of the synthesized compounds showed good to moderate activity against a panel of standard Gram-positive and Gram-negative bacteria and fungal strains. The compounds 7i and 7v exhibited significant activity, with a MIC value of 2.0-6.0 µg/mL against a panel of Gram-positive and Gram-negative bacteria. These compounds also showed activity against Candida albicans, with a MIC value of 4.0 µg/mL. A correlation of the antimicrobial activity with calculated lipophilicity values (C log P) is also presented.

A sociodemographic study of multidrug resistant tuberculosis cases from DOTS clinics of Kolkata.

This cross-sectional, observational study was undertaken to identify rifampicin and INH resistant mycobacteria in the sputum samples of category II treatment failure tuberculosis patients from three 'DOTS' clinics of Kolkata (Ward No 15, 33, 79 of Kolkata Municipal Corporation). Conventional method of culture in solid and liquid media followed by sensitivity testing to rifampicin and INH were done. Age, sex, economic status, nutrition, history of contact, living condition, etc, were taken into consideration. Out of 66 category II treatment failure tuberculosis cases from 3 DOTS clinics, 45 (68.2%) were found to be multidrug resistant (MDR). Among them, 68.9% were male patients and 55.6% were in the economically productive age group. Most of them (73.3%) were malnourished (BMI < 18.5); 62.2% of patients belonged to 'below poverty line' (BPL) category and 82.2% patients lived in overcrowded room. Majority of them (82.3%) either had past history of tuberculosis or history of contact or both.This study also shows that apart from the biological reasons of drug resistance, the sociodemographic and economic factors are no less important for the spread of drug resistant tuberculosis.

The silent burden of anemia in school age children: a community based study in West Bengal.

BACKGROUND: Under nutrition and anemia are common co-morbidities in school age children. Due to transition in dietary habits in developing countries, a paradoxical finding of coexistence of anemia and normal/over nutrition is also a cause of concern. OBJECTIVE: > T o assess the nutritional status and prevalence of anemia among school age children (6 - 16 years) residing in rural and urban areas of a district of West Bengal and also to find out the association between weight status, measured as Body Mass Index(BMI) and anemia. MATERIALS AND METHODS: Age, height & weight were measured in 86 rural and 86 urban school age (6 -16 years) children in rural and urban field practice areas of Midnapore Medical College. Their blood was estimated for haemoglobin concentration. RESULTS: Overall prevalence of anemia was 80.2%, and not significantly different between the rural (83.7%) and urban (76.7%) participants and across the genders both in rural (86.4% versus 80.9%) and urban (85.7% versus 72.4%) areas. Thinness was observed to be higher in urban area (48.8% versus 41.9%). However, severe thinness was higher in rural area (18.5% versus 13.9%). Significantly, higher proportion of boys revealed severely low BMI compared to girls in both rural (33.3% versus 4.5%) and urban (17.2% versus 7.1%) areas with no significant differences between the prevalence of anemia across the grades of underweight and normal nutritional status. CONCLUSIONS: Poor nutritional status and anemia are still, taking heavy toll and new program strategies are needed, particularly those that improve the overall nutrition status of children.

Study of serum electrolytes in pregnancy induced hypertension.

Hypertension complicating pregnancy is an important cause of foetal and maternal mortality and morbidity. Serum sodium, potassium, calcium and magnesium were evaluated in cases with gestational hypertension and pre-eclampsia and compared with normotensive pregnant women of corresponding period of gestation. Taking urinary micro-albumin creatinine ratio as standard to predict pregnancy induced hypertension, the role of serum electrolytes to predict the same was studied. A total of 200 patients were evaluated. There were no change in serum sodium and potassium in gestational hypertensive cases as well as in pre-eclamptic cases when compared with control group. Similar results were obtained when serum calcium level was evaluated. Mean serum magnesium level (1.53 +/- 0.29 mg/dl) was significantly low in cases of pre-eclampsia, while it was 1.79 +/- 0.25 mg/dl in patients with gestational hypertension and 2.19 +/- 0.2 mg/dl in control group. Patients having gestational hypertension along with increased micro-albumin creatinine ratio had significantly low mean magnesium level (1.68 +/- 0.1 mg/dl) when compared with gestational hypertensive patients with normal microalbumin creatinine ratio (serum magnesium level 1.87 +/- 0.1 mg/dl).

Rapid identification of mycobacterium species with the aid of multiplex polymerase chain reaction (PCR) from clinical isolates.

Mycobacteria are aerobic, nonspore forming, non-motile,single-cell bacteria.Of more than 40 currently recognized species of mycobacteria, Mycobacterium tuberculosis, the causative agent of human TB is the commonest pathogen for pulmonary and extra pulmonary tuberculosis cases. The other members of the Mycobacterium tuberculosis complex (MTC) or the nontubercular mycobacterium (NTM) produces similar diseases which cannot be differentiated from tuberculosis by clinical symptoms and signs. But this differentiation is important as the chemotherapy varies widely according to the strain of mycobacterium. The burden of morbidity and mortality of tuberculosis is rapidly growing worldwide, particularly with the HIV/AIDS epidemic. The strain identification of Mycobacterium remains a cumbersome, labor intensive and expensive procedure, which requires 3 to 12 weeks of time. The conventional methods of strain identification lack proper standardization and precise diagnosis. The prime objective of this study is to overcome these problems.A multiplex PCR using 3 amplicons of 165,365, and 541 base pair target sequences was done with a total number of 165 clinical isolates of suspected Koch's patients. Strain identification was compared both by conventional methods and multiplex PCR. The results of the study show that this multiplex PCR is supposed to be less complicated, less time consuming, cost-effective and superior to the conventional methods. It is also applicable for culture negative samples where strain identification is not possible by conventional approach.

A hospital-based study to evaluate the incidence pattern of group A streptococcal throat infections from different age group patients.

Streptococcus pyogenes(group A) is a major pathogen capable of causing a wide range of diseases in different age group of people. In this study 100 patients were selected who presented with the complaint of sore throat. All the patients were divided in four age groups. Streptococcus pyogenes colonies were confirmed on the basis of beta-haemolysis, bacitracin sensitivity test, and latex agglutination test for group A. Out of a total of 100 samples, 42 were confirmed as group A streptococcus. From this study, it has been observed that all age groups, with maximum occurrence in 5-15 years age group, were suffering from group A streptococcal pharyngitis. Therefore every case of sore throat especially affecting children should be investigated to detect the causative agent for initiation of proper therapy so that the more serious outcome like acute rheumatic fever (ARF) and acute glomerulonephritis (AGN) can be prevented.

Prevalence of hypothyroidism and importance of cholesterol estimation in patients suffering from major depressive disorder.

The coexistence of hypothroidism and depression is already reported and both of these conditions are known to share some common clinical symptoms. Serum cholesterol level is known to be altered in either condition considered separately. But, no report is obtained regarding serum cholesterol level when both the conditions coexist. In this study, 78 patients (61 females and 17 males, age ranged 19 to 67 years) suffering from major depressive disorder were included. Serum T3, T4, TSH and cholesterol levels were estimated in all of them. Sixty-two patients were found to be euthyroid and 16 patients (11 females, 5 males) were found to be hypothyroid. Among female patients, 6 had subclinical hypothyroidism and 5 had overt hypothyroidism. Among male patients 3 had subclinical hypothyroidism and 2 had overt hypothyroidism. The overall prevalence of hypothyroidism in major depressive disorder was estimated as 20.5%. Mean serum cholesterol level in 62 euthyroid patients was found to be 150.9% +/- 16 mg% and that of 16 hypothyroid patients to be 190.7 +/- 12 mg% showing a significant difference (p < 0.01). Thus estimation of cholesterol in major depressive disorder patients may give an idea regarding their thyroid status and vice-versa.

Development and application of a new efficient and sensitive multiplex polymerase chain reaction (PCR) in diagnosis of leprosy.

India contributes about 80% of the global leprosy case load including case of fresh infection and reinfection. Due to lack of gold standard, diagnosis is done mainly based on routine clinical signs and symptoms, smear and histopathological evidences. There is a lot of lacunae in early confirmatory diagnosis in terms of sensitivity and specificity, especially in paucibacillary tuberculoid type. Moreover, the classification of different classes of leprosy is very important for selection of proper therapeutic schedule. Hence this study was undertaken to develop a multiplex polymerase chain reaction for the diagnosis and strain differentiation of M leprae. A multiplex polymerase chain reaction was developed using the primers R1 and R2 (a) amplifying 372bp DNA target from a repetitive sequence of M leprae and this repetitive sequence (372bp) that was used as a target DNA for amplification was reported to be specific for M leprae was not present in 20 mycobacterium species other than M leprae and primers TTCA and TTCB (b) amplifying (201bp) DNA target of variable sizes from the regions flanking TTC repeats of M leprae genome. This multiplex polymerase chain reacton developed in our laboratory revealed that the number of repeats at each locus might be variable among M leprae but they are found mostly in multibacillary (as the bacterial load is higher in multibacillary) type.