Polymerase Chain Reaction Restriction Analysis Research Articles

Direct Molecular Characterization of Acid-Fast Bacilli Smear of Nontuberculosis Mycobacterium Species Causing Pulmonary Tuberculosis in Guna Yala Region, Panama.

Mycobacterium tuberculosis (MTB) stands out as the main causative agent of pulmonary tuberculosis (TB). However, nontuberculous mycobacteria (NTM) species also have the potential to infect and cause TB in susceptible individuals. The objective of this study was to identify NTM species that cause public health problems in remote areas. The study was carried out using 105 sputum smears obtained from patients from the Guna Yala Region of Panama with clinical signs suggestive of TB. DNA was extracted from sputum smears. Nontuberculous mycobacteria and MTB were characterized using polymerase chain reaction restriction analysis (hsp65, rpob) and an evaluation of 24-mycobacterial interspersed repetitive units-variable number of tandem repeats loci. Twenty-six Mycobacterium species were characterized; 19 (18%) were identified as MTB, and 7 (6.7%) were identified as NTM (four M. avium complex, two M. haemophilum, one M. tusciae). These results suggest that at least one in five cases of pulmonary TB among this population is caused by an NTM. Thus, identifying the bacteria causing pulmonary disease is key even in remote regions of the world where standard diagnosis and culture are not available. Strengthening the laboratory capacity within the Guna Yala Region is needed to identify NTM infections promptly.

Are We Neglecting Nontuberculous Mycobacteria Just as Laboratory Contaminants? Time to Reevaluate Things.

Objectives Nontuberculous mycobacteria (NTM) incidences are on the rise worldwide, including the tuberculosis endemic areas. They should be identified rapidly to the species level and should be carefully differentiated as contamination, colonization, or disease. This study was aimed at determining the prevalence and clinicoepidemiological profile of mycobacteriosis cases. Materials and Methods Cultures were made on liquid and solid media. NTM were identified by polymerase chain reaction (PCR) restriction analysis (PRA) and gene sequencing. Data was analyzed using Epi-info 7. Results Out of the 1042 processed specimens, 16% were positive for M. tuberculosis complex and 1.2% for clinically significant NTM. M. intracellulare was the commonest species isolated. NTM were treated mainly on outdoor basis (92%), involving more extrapulmonary system (62%) and higher age-group of 41-60 years (69%). No significant factor was seen to be associated clinically, radiologically, and biochemically with the NTM infections. Conclusions Our study highlights the importance of early diagnosis and differentiation among Mycobacterium tuberculosis and NTM so that these NTM are not underestimated in routine diagnostic procedures merely as environmental or laboratory contaminants.

PIRA-PCR for Detection of Fusarium fujikuroi Genotypes with Carbendazim-Resistance Alleles.

Carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide, has been used to control rice bakanae disease, caused by Fusarium fujikuroi (teleomorph: Gibberella fujikuroi), for decades in China. Previous research revealed that point mutations (E198V, GAG to GTG at codon 198, and F200Y, TTC to TAC at codon 200) of the β2-tubulin gene conferred resistance of F. fujikuroi to MBC. In this study, primer-introduced restriction analysis polymerase chain reaction (PIRA-PCR) was developed to determine genotypes with resistance of F. fujikuroi to MBC. A PCR template of each strain was created by an outer primer pair. Fragments with 177 bp (for mutation at codon 235) and 146 bp (for E198V) were amplified by nested PCR, with two inner primer pairs designed and synthesized according to the nucleotide sequence of β2-tubulin for further enzyme digestion validation, respectively. AccII and PmaCI restriction enzyme recognition sites were introduced artificially by inner primers to differentiate MBC-sensitive and -resistant strains, respectively. The sensitivity of each strain to MBC was indirectly determined by analyzing electrophoresis patterns of the resulting amplified fragments after simultaneous digestion by both AccII and PmaCI. PIRA-PCR produced the same result as conventional methods in 6% of the time. PIRA-PCR is a sensitive and effective method for genotyping resistance alleles of F. fujikuroi strains to MBC.

Detection of global hypermethylation in well-differentiated thyroid neoplasms by immunohistochemical (5-methylcytidine) analysis.

While global hypomethylation of DNA has been found in several malignancies, studies on thyroid tumours have shown controversial results using different techniques. To help resolve this issue, we assessed methylation status using two different techniques in papillary thyroid carcinomas (PTC) and follicular adenomas (FA) and carcinomas (FTC), comparing adjacent non-neoplastic thyroid tissue. A series of 15 FA, 18 FTC and 17 PTC were assessed by: (1) measurement of methylation levels of long interspersed nuclear elements (LINE-1) using a combined bisulfite restriction analysis polymerase chain reaction protocol and (2) immunostaining with an anti-5-methylcytidine antibody that detects methylated DNA regardless of the DNA sequence. Immunostaining was scored by image analysis. Methylation levels of LINE-1 in FA, FTC and PTC were not significantly different from adjacent normal tissue. There was no significant difference in methylation levels of LINE-1 between FA, FTC and PTC (p = 0.44). By immunohistochemical staining for methylation, the 5-methylcytidine score was significantly higher in tumours than in normal tissue counterparts, for FA (p < 0.001), FTC (p = 0.04) and PTC (p = 0.02). PTC showed the highest 5-methylcytidine expression amongst all tumours which was significantly different from FTC (p = 0.015), but not FA (p = 0.09). There was no correlation in methylation level between LINE-1 and 5-methylcytidine scores for each group and overall. Well-differentiated thyroid neoplasms (FA, FTC and PTC) were not found by two independent methods to undergo global hypomethylation as part of an oncogenic sequence from normal tissue to carcinoma. Instead, hypermethylation was detected in all types of tumours, implying that this epigenetic event may contribute to oncogenic development of thyroid neoplasms (both benign and malignant).

Primer-Introduced Restriction Analysis Polymerase Chain Reaction Method for Non-Invasive Prenatal Testing of β-Thalassemia

We have developed a new method for non-invasive prenatal testing (NIPT) of paternally inherited fetal mutants for β-thalassemia (β-thal). Specially designed primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) were used to detect four major mutations [IVS-II-654, HBB: c.316-197C > T; codon 17 (A > T), HBB: c.52A > T; −28 (A > G), HBB: c.-78A > G and codons 41/42 (–TTCT), HBB: c.126_129delCTTT] causing β-thal in China. The PIRA-PCR assay was first tested in a series of mixed DNA with different concentrations and mixed proportions. Subsequently, this assay was further tested in 10 plasma DNA samples collected from pregnant women. In the DNA mixture simulation test, the PIRA-PCR assay was able to detect 3.0% target genomic DNA (gDNA) mixed in 97.0% wild-type gDNA isolated from whole blood. For plasma DNA testing, the results detected by PIRA-PCR assay achieved 100.0% consistency with those obtained from the amniocentesis analysis. This new method could potentially be used for NIPT of paternally inherited fetal mutants for β-thal.

Molecular characterisation of clinical and environmental isolates of Mycobacterium kansasii isolates from South African gold mines.

Mycobacterium kansasii (M. kansasii) is a major cause of non-tuberculous mycobacterial pulmonary disease in the South African gold-mining workforce, but the source of infection and molecular epidemiology are unknown. This study investigated the presence of M. kansasii in gold and coal mine and associated hostel water supplies and compared the genetic diversity of clinical and environmental isolates of M. kansasii. Five M. kansasii and ten other potentially pathogenic mycobacteria were cultured mainly from showerhead biofilms. Polymerase chain reaction-restriction analysis of the hsp65 gene on 196 clinical and environmental M. kansasii isolates revealed 160 subtype I, eight subtype II and six subtype IV strains. Twenty-two isolates did not show the typical M. kansasii restriction patterns, suggesting that these isolates may represent new subtypes of M. kansasii. In contrast to the clonal population structure found amongst the subtype I isolates from studies in other countries, DNA fingerprinting of 114 clinical and three environmental subtype I isolates demonstrated genetic diversity amongst the isolates. This study demonstrated that showerheads are possible sources of M. kansasii and other pathogenic non-tuberculous mycobacterial infection in a gold-mining region, that subtype I is the major clinical isolate of M. kansasii strain and that this subtype exhibits genetic diversity.

Bias toward a 1:1 ratio in primer-introduced restriction analysis PCR: mechanism and minimization.

Primer-introduced restriction analysis is widely used in molecular genetics. However, several studies have reported inconsistent data regarding sequencing, mainly among heterozygous samples. This discrepancy may be related to the bias towards a 1:1 ratio typically observed in heterozygous digestion products. In this study, we investigated the mechanism and minimization of this observed bias. Three mismatched polymerase chain reaction (PCR) models were analyzed by testing different PCR conditions and reaction mixtures. For EPHX1 gene rs1051740 single-nucleotide polymorphism PCR, DNA concentration, denaturation and elongation time, annealing temperature, and cycle number significantly influenced product ratios. For SERPINA1 gene PIMmalton deletion (ΔPhe52) and CHRNA3 gene rs1051730 single-nucleotide polymorphism PCRs, significant bias fluctuations were observed only for the annealing temperature and cycle number conditions. The relevance of these results to the amplification efficiency parameter is discussed. Rather than reducing the observed bias, our data provide evidence of a counterbalance for preferential amplification, depending on cycle number, annealing temperature, and amplification efficiency alteration. Our results are relevant for application to primer-introduced restriction analysis PCR assays.

Clinical isolates of Mycobacterium abscessus in Guangzhou area most possibly from the environmental infection showed variable susceptibility

Background Mycobacterium abscessus (M. abscessus) can cause a variety of human infections, involving the lung, skin and soft tissues, and is generally believed to be acquired from environmental sources. The aim of this study was to investigate the molecular diversity and antibiotic susceptibility of M. abscessus isolates as the basis for strategies to improve control and management of infection. Methods Seventy M. abscessus isolates from patients attending the Guangzhou Thoracic Hospital were identified from 2003 to 2005 by biochemical tests, gas chromatography, polymerase chain reaction (PCR)-restriction analysis (PRA) of heat shock protein gene hsp65, and sequencing of the quinolone resistance determining regions (QRDRs) of gyrA. Susceptibilities to six antibiotics were determined by micro-broth dilution. Isolates were genotyped using randomly amplified polymorphic DNA (RAPD) analysis. Results Most isolates (63/70; 90%) were susceptible to amikacin but rates of susceptibility to other antibiotics varied from moderate, clarithromycin (60%) and imipenem (43%), to low for ciprofloxacin and ofloxacin (3%), and 87% of isolates had intermediate susceptibility to cefoxitin. RAPD analysis showed that the 70 clinical isolates displayed 69 unique RAPD patterns. Conclusions The high genetic diversity of isolates suggests that they are not transmitted from person to person but, presumably, are acquired independently from environmental sources. M. abscessus isolates displayed variable levels of susceptibility to all antibiotics tested, other than amikacin, indicating a need for routine susceptibility testing to guide treatment.

Are we overlooking infections owing to non-tuberculous mycobacteria during routine conventional laboratory investigations?

A large number of potentially pathogenic non-tuberculous mycobacteria (NTM) encountered in the clinical laboratory makes it necessary to identify their species to ensure appropriate treatment. However, labor-intensive conventional methods of speciation are not used in every laboratory, and hence NTM infections are often ignored. Polymerase chain reaction (PCR) restriction analysis (PRA) was applied in this study for early identification and speciation of mycobacterial species on 306 cultures of acid-fast bacilli isolated from patients suspected of suffering from tuberculosis. Mycobacterium tuberculosis was identified in 85.6% of the isolates. The NTM isolated most commonly was Mycobacterium kansasii/gastri group (3.5%), followed by Mycobacterium fortuitum (3.2%). Four of the M. fortuitum were grown from cultures obtained on the same day, but from samples from different patients and were probably laboratory contaminants. Mycobacterium intracellulare and Mycobacterium avium were identified in 2.94% and 2.28% of the isolates, respectively. Three isolates of M. avium and two isolates of M. intracellulare were obtained in repeated cultures from sputum samples of the same patients and were thus pathogenic. A single isolate of Mycobacterium abscessus was obtained from a breast abscess. A rare pathogen Mycobacterium phocaicum was isolated from one patient with epididymitis. However, whether it was the causative agent of epididymitis in this patient remains doubtful. The results of this study highlight the importance of speciation of mycobacteria for appropriate diagnosis and the importance of including molecular assays to augment conventional methods of diagnosis of mycobacterial diseases for rapid identification of NTM so that these potential pathogens are not overlooked in routine diagnostic procedures.

Detection and Molecular Characterization of Boscalid-Resistant Botrytis cinerea Isolates from Strawberry.

Botrytis cinerea isolates (n = 122) were collected from strawberry fields located in northern Greece during a 3-year period (2008-10) and tested for their sensitivity to the succinate dehydrogenase inhibitor boscalid. Sensitivity measurements showed three distinct phenotypes consisting of isolates highly sensitive (fungicide concentration causing inhibition of germ tube growth by 50% [EC50 values] of 0.05 to 0.21 μg ml-1), moderately resistant (EC50 values of 1.37 to 7.79 μg ml-1), or highly resistant (EC50 values of >50 μg ml-1) to boscalid. Sequence analysis of the sdhB gene revealed five mutations leading to amino acid substitutions in the SdhB subunit in isolates moderately resistant and highly resistant to boscalid. Three moderately resistant isolates showed a nucleotide change from A to T at codon 230, resulting in an asparagine to isoleucine (N230I) substitution. Several moderately resistant isolates showed a nucleotide change from C to T at codon 272, resulting in a substitution from histidine to arginine (H272R) whereas, in another set of isolates, a nucleotide change from A to G was found at the same codon, leading to a substitution from histidine to tyrosine (H272Y). One highly resistant isolate had a nucleotide change from A to T at codon 272, leading to a substitution from histidine to leucine (H272L), whereas, in three other highly resistant isolates, a double nucleotide change from CC to TT was observed at codon 225, resulting in a substitution from proline to phenylalanine (P225F). To facilitate rapid detection of these mutations associated with resistance to boscalid, a primer-introduced restriction analysis polymerase chain reaction was developed. The method was successfully applied to the moderately and highly resistant subpopulations and showed that the H272R mutation was predominant with relative frequencies of 28.5, 37.5, and 30% during 2008, 2009, and 2010, respectively. In contrast, the H272L mutation was detected at a frequency of 2.5% only in the 2009 population, whereas the P225F mutation was detected at a frequency of 7.5% only in the 2010 population.

Molecular epidemiological analysis of Mycobacterium kansasii isolates

To make molecular epidemiological analysis of Mycobacterium kansasii (M. kansasii) isolates. We examined 174 M. kansasii isolates from clinical samples of patients at National Hospital Organization Kinki-chuo Chest Medical Center from June 1, 2002 to August 31, 2005 by polymerase chain reaction (PCR) -restriction analysis (PRA) of the heat shock protein (hsp) 65 gene (hsp65-PRA), sequencing (ITS, 16S-23S internal transcribed spacer, and hsp65 for discrepant case between hsp65-PRA and ITS sequence), pulsed-field gel electrophoresis (PFGE), and restriction fragment length polymorphism (RFLP) with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe of genomic DNA. Of the 174 M. kansasii isolates, 170 strains were classified as M. kansasii type I using hsp65-PRA, while two isolates belonged to type II and one each isolate to type IIb and VI, respectively. Although the ITS sequence of these isolates also identified the same region of polymorphism by hsp 65-PRA, only type II b might be revealed atypical type II, a transitional type from typical type II to intermediate type I by hsp65 sequence. The polymorphic patterns by RFLPs with MPTR and IS1652 probe were shown specific for each homogeneous cluster by hsp 65-PRA. In addition, 159 isolates were recognized the same common pattern A by PFGE analysis. In contrast, the rest 15 isolates revealed significant polymorphism within 11 isolates of type I, and 4 isolates among type II, IIb, and VI. We verified the M. kansasii genotype I was predominant, with the same pattern of major worldwide type regions, and reflected a very tight clonal structure. Type I was furthermore indicated recognition of subtypes by PFGE analysis.

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.

Isolation ofMycobacterium tuberculosisfrom CaptiveAteles paniscus

An adult female red-faced black spider monkey (Ateles paniscus), housed for 2 years in the Parque Estoril Zoo in São Paulo, Brazil, showed apathy. Clinical examination revealed discrete emaciation, swelling and induration of lymph nodes, and presence of a mass in the abdominal cavity. Therapies with enrofloxacin, azithromycin, and ceftiofur were ineffective. The animal died after 6 months. Necropsy and histopathology confirmed granulommas in lymph nodes, parietal and visceral pleura, lungs, liver, spleen, and kidneys. Acid-fast bacilli were isolated and identified as Mycobacterium tuberculosis by polymerase chain reaction restriction analysis and Spoligotyping techniques. The zoo personnel and other animals that had had contact with the infected primate were negative to tuberculosis diagnostic procedures, such as sputum exam (baciloscopy) and thorax radiography. It was impossible to determine whether the infection occurred before or after the arrival of the animal to the Parque Estoril Zoo. This is the first report of M. tuberculosis infection in Ateles paniscus, a neotropical primate.

Doença pulmonar por Mycobacterium tuberculosis e micobactérias não-tuberculosas entre pacientes recém-diagnosticados como HIV positivos em Moçambique, África

Mycobacteriosis is frequently diagnosed among HIV-infected patients. In Mozambique, where few patients are under antiretroviral therapy and the prevalence of tuberculosis is high, there is need for better characterization of mycobacteria at the species level, as well as for the identification of patterns of resistance to antituberculous drugs. We studied a sample of 503 HIV-infected individuals suspected of having pulmonary tuberculosis. Of those 503, 320 tested positive for mycobacteria through sputum smear microscopy or culture of bronchoalveolar lavage fluid. Acid-fast bacilli were observed in the sputum of 73% of the individuals presenting positive cultures. Of 277 isolates tested, only 3 were nontuberculous mycobacteria: 2 were identified as Mycobacterium avium and one was identified as M. simiae. Strains initially characterized as M. tuberculosis complex through polymerase chain reaction restriction analysis (PRA) of the hsp65 gene were later confirmed as such through PRA of the gyrB gene. Among the M. tuberculosis isolates, resistance patterns were as follows: to isoniazid, 14%; to rifampin, 6%; and multidrug resistance, 5%. Previously treated cases showed significantly higher rates of resistance to first-line antituberculous drugs. The most common radiological pattern was interstitial infiltrate (in 67%), followed by mediastinal lymph node enlargement (in 30%), bronchiectasis (in 28%), miliary nodules (in 18%) and cavitation (in 12%). Patients infected with nontuberculous mycobacteria presented clinical profiles indistinguishable from those of other patients. The median CD4 lymphocyte count in this group was 134 cells/mm(3). There is a strong association between tuberculosis and AIDS in Mozambique, as expected in a country with a high prevalence of tuberculosis. Although drug resistance rates are high, the isoniazid-rifampin regimen continues to be the appropriate choice for initial therapy.

Detection of LINE-1s hypomethylation in oral rinses of oral squamous cell carcinoma patients

This study aimed to (i) investigate long interspersed nuclear element-1 (LINE-1) methylation levels of oral squamous cell carcinomas (OSCCs), the major type of oral malignancies; and (ii) investigate whether the hypomethylation of LINE-1s can be detected in oral rinses of OSCC patients. The combined bisulfite restriction analysis polymerase chain reaction (PCR) of LINE-1s (COBRALINE-1) was used. We found that tissues from OSCC specimens had lower methylation levels of LINE-1s than cells collected from the oral rinses of normal volunteers. Interestingly, cells collected from oral rinses of OSCC patients also revealed hypomethylated LINE-1s at the same level as OSCC tissues. There was no difference in the level of hypomethylation due to stages, locations, histological grades, and history of betel chewing, smoking and/or alcohol consumption. In conclusion, OSCCs possessed global hypomethylation and this alteration could be detected from oral rinses of OSCC patients by a simple PCR technique, COBRALINE-1. Therefore, COBRALINE-1 of oral rinses may be applied for non-invasive detection of oral malignancies.

LINE-1 methylation status of endogenous DNA double-strand breaks

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

Identification of Mycobacterium marinum 65 kD heat shock protein gene by polymerase chain reaction restriction analysis from lesions of swimming pool granuloma

Nontuberculous mycobacterium (NTM) had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum (M. marinum) are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction (PCR) was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein (hsp) 65 kD gene. The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis (PRA) identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.

Examination of Gould’s modified S1 (mS1) selective medium and Angle’s non-selective medium for describing the diversity of Pseudomonas spp. in soil and root environments

Studies on the diversity of environmental culturable Pseudomonas populations are dependent on the isolation procedure. This procedure includes the use of selective media which may influence the recovery of strains and thus the diversity described. In this study, we assessed the use of two agar isolation media for describing the diversity of soil- and root-inhabiting Pseudomonas associated with the perennial grass Molinia coerulea. A total of 382 Pseudomonas strains were recovered on either non-selective Angle’s medium, or on Gould’s modified S1 (mS1) Pseudomonas-selective medium. Their diversity was assessed by restriction analysis of PCR (polymerase chain reaction)-amplified 16S–23S rDNA internal transcript spacer sequences. The comparison of mS1- and Angle-recovered populations showed that the use of mS1 selective medium led to an underestimation of both Pseudomonas counts and diversity, especially in the soil environment.

Leishmania donovani: Intraspecific Polymorphisms of Sudanese Isolates Revealed by PCR-based Analyses and DNA Sequencing

El Tai, N. O., El Fari, M., Mauricio, I., Miles, M. A., Oskam, L., El Safi, S. H., Presber, W. H., and Schönian, G. 2001. Leishmania donovani: Intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Experimental Parasitology97, 35–44. Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.