Polymerase Chain Reaction Products Of Size Research Articles

Identification of Candida Isolates from Cancer Patients Using Multiplex PCR

Aim of this study was to identify the Candida isolates from Cancer Hospital Maharagama, Sri Lanka by using Multiplex PCR (polymerase chain reaction) method. In this study, Multiplex PCR was used to identify Candida isolates from cancer patients at Apeksha Hospital, Sri Lanka. In multiplex PCR, 18 specific primers (nine primer pairs) were separated into three groups for multiplex PCR (PS I, PS II and PS III). Different sized PCR products corresponding to each Candida species were produced by PCR using the primer mixes. Among 52 Candida isolates, 49 were identified using the multiplex PCR procedure. Candida tropicalis was found to be the most prevalent species (38%), followed by Candida parapsilosis (31%), Candida albicans (13%), Candida glabrata (8%) and Candida krusei (4%). The study concludes that multiplex PCR is a better approach for the identification of Candida species and recommends the use of it for clinical and diagnostic purposes and for research purposes.

Mutational analysis of exon 8 and exon 14 of ATP7B gene in Bangladeshi children with Wilson disease.

It was a cross sectional observational study. The study was conducted at the Department of Paediatric Gastroenterology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh from January 2017 to June 2018. A total of 37 patients diagnosed with WD were enrolled for the study. Venous blood (about 3 mL) was drawn aseptically from each patient into tube containing ethyline diamine tetraacetic acid (EDTA) and preserved at -30°C for long-term preservation. The peripheral blood leukocytes of the patients and genomic DNAs were extracted. Exons 14 and 8 of ATP7B and their associated splice-site junctions were amplified by the polymerase chain reaction (PCR). The size and quantity of PCR products were verified by electrophoresis in 1.5% (w/v) agarose gel. 74 (37 × 2) PCR products were sent for Sanger Sequencing. The sequences were analyzed by Chromas version 2.6.6 software and the nucleotide blast was done by National Center for Biotechnology Information (NCBI) nucleoblast. Finally, the sequences were analyzed using AB Applied Bio systems and were matched with the reference sequences using MEGA software. In this study, a single novel homozygous mutation pLeu.1071Val in the exon 14 was found in every (100%) studied child clinically diagnosed with WD. Heterozygous mutation p.Gly1061Glu in exon14 was also found in 6 patients (11%) with WD, which is one of the common mutations in this disease. In exon 8, p.Arg778Leu mutation was detected in one patient (2.7%), which is common in the Chinese and the South Asian populations and was heterozygous. Two novel heterozygous missense mutations p.K785R (2.7%) and p.S744F (2.7%) were also found in two other children in the exon 8. We found three novel mutations in Bangladeshi children with WD, one of which may be tagged as founder mutation for Bangladeshi population. This finding indicates the necessity to study the mutation profiles of the whole ATP7B gene in our population for risk prediction. A further large-scale study will help in the development of a Mutational Data Base of Bangladeshi population with WD.

Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-3 Alpha and Beta from Diverse Geographic Areas of Thailand.

Malaria is parasitic disease cause by Plasmodium infection. In Thailand, coinfections of Plasmodium vivax and Plasmodium falciparum are commonly found. P. vivax infection has been increasing in the past decade. The objective of this study was to investigate the genetic diversity patterns of P. vivax merozoite surface protein 3 (PvMSP-3) genes in a total of 450 isolates collected from Thai-neighboring border areas during two different periods (2009-2014 and 2015-2016) using polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Three major types of PvMSP-3α (A, B, and C) and PvMSP-3β (A, B, and C) were detected based on PCR product size. In total, 45 and 23 alleles of PvMSP-3α and 41 and 30 alleles of PvMSP-3β with different frequencies of samples were distinguished from the first and second period, respectively. Our results strongly indicate genetic diversity patterns of PvMSP-3 in isolates from the second period, especially in samples from the Thai-Myanmar border area. These two polymorphic genes could be used as molecular epidemiologic markers for genotyping P. vivax isolate in Thailand.

Nitrilase gene detection and nitrile metabolism in two bacterial strains associated with waste streams in Lagos, Nigeria

BackgroundThe use of nitrile compounds is usually high, particularly in chemical industries, which calls for serious concern because of their relevance to the environment. The essential role of nitrilases in the bioremediation of harmful nitriles from environmental wastes cannot be overemphasized. The study aimed to unveil the biodegradative potentials of bacterial strains associated with the degradation of nitrile pollutants.MethodsBacterial strains capable of utilizing glutaronitrile as the sole source of carbon and nitrogen were isolated from solid waste leachates by a selective enrichment culture technique. The test organisms were grown in mineral salts medium (MSM), and the metabolic products were determined using gas chromatography-flame ionization detection (GC-FID). The nitrilase gene was amplified by polymerase chain reaction (PCR) and by using appropriate primers.ResultsThe growth studies showed that the test organisms grew on the two nitriles. The doubling times of 12.16 d and 9.46 d (specific growth rate, µ=0.082 d−1, 0.106 d−1) were obtained for each pure culture of Bacillus sp. srain WOD8 and Corynebacterium sp. srain WOIS2 on glutaronitrile (as single substrate), respectively. While the same strains had doubling times of 11.11 d and 10.00 d (µ=0.090 d−1, 0.100 d−1) on benzonitrile (as single substrate). However, the mixed culture (comprising the two strains) had doubling times of 7.40 d and 7.75 d (µ=0.135 d−1, 0.129 d−1) on glutaronitrile (as single and mixed substrates), respectively. While doubling times of 8.09 d and 8.71 d (µ=0.124 d−1, 0.115 d−1) were obtained for the same mixed culture on benzonitrile (as single and mixed substrates). Based on gas chromatographic analysis, the residual glutaronitrile concentrations at day 16 for strains WOD8 and WOIS2 were 35.77 g L−1 (72.2%) and 9.30 g L−1 (92.5%), respectively, whereas the residual benzonitrile concentrations for the same strains were 27.39 g L−1 (78.8%) and 13.79 g L−1 (89.2%), respectively. For the mixed culture, residual glutaronitrile and benzonitrile concentrations at day 16 were 13.40 g L−1 (88.5%) and 10.42 g L−1 (91.5%), respectively, whereas for the mixed substrates (glutaronitrile and benzonitrile), 7.21 g L−1 (91.7%) and 4.80 g L−1 (94.2%) of residual glutaronitrile and benzonitrile concentrations were obtained by the same consortium. The gene for nitrilase involved in nitrile degradation was detected in the genome of the bacterial strains. The amplified nitrilase gene gave PCR products of sizes 1400 bp and 1000 bp, as expected for strains WOD8 and WOIS2, respectively. 4-Cyanobutyric acid (4CBA), glutaric acid (GA), and benzoic acid (BA) were obtained as metabolites following nitrile degradation in vitro.ConclusionThese results revealed that strains WOD8, WOIS2 and the mixed culture (consisting of the two strains) have proven to have the capacity to metabolize nitriles (glutaronitrile and benzonitrile) as the carbon and nitrogen sources. However, the mixed culture had higher nitrile degradation rate as compared to each pure culture of the two test organisms. These results also provide insight into the evolutionary genetic origin of a nitrilase gene that encodes an enzyme that catalyzes nitrile degradation in these strains. Hence, the bacterial strains that harbor this kind of gene may be used as promising biological agents for the remediation of sites polluted with nitriles, thereby opening new perspectives for encouraging data for a bioremediation bioprocess.

Remarkable genetic diversity of Trypanosoma cruzi and Trypanosoma rangeli in two localities of southern Ecuador identified via deep sequencing of mini-exon gene amplicons

BackgroundTrypanosoma cruzi, the causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas disease prevalence and transmission dynamics. Trypanosoma cruzi possesses high molecular heterogeneity: seven discrete typing units (DTUs) are currently recognized. In Ecuador, T. cruzi TcI and T. rangeli KP1(-) predominate, while other genetic lineages are seldom reported.MethodsInfection by T. cruzi and/or T. rangeli in different developmental stages of triatomine bugs from two communities of southern Ecuador was evaluated via polymerase chain reaction product size polymorphism of kinetoplast minicircle sequences and the non-transcribed spacer region of the mini-exon gene (n = 48). Forty-three mini-exon amplicons were also deep sequenced to analyze single-nucleotide polymorphisms within single and mixed infections. Mini-exon products from ten monoclonal reference strains were included as controls.ResultsTrypanosoma cruzi genetic richness and diversity was not significantly greater in adult vectors than in nymphal stages III and V. In contrast, instar V individuals showed significantly higher T. rangeli richness when compared with other developmental stages. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 8 T. cruzi infections (23.5%) and 25 T. cruzi + T. rangeli co-infections (73.5%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep sequencing detected TcIV sequences in nine samples; this DTU had not previously been reported in Loja Province.ConclusionsOur data indicate that deep sequencing allows for better parasite identification/typing than amplicon size analysis alone for mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (c.28 km apart), suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.

Detection of Aspergillus fumigatus by Polymerase Chain Reaction (PCR)

Aspergillosis refers to fungi infections of the respiratory tract caused by Aspergillus species, especially Aspergillus fumigatus. Infection of A. fumigatus was increased in the last few years due to either resistances to antibiotics or the influence of other factors such as other fungal infections. The present study aimed to review the impact of Aspergillus fumigatus in Aspergillosis cases, and study the role of Singleplex PCR for amplification of ITS1, ITS4 of rRNA gene in the detection of fungal isolate. In this study, One hundred sputum samples were collected from patients admitted to the specialize chest and respiratory diseases center / Baghdad who were suffering from respiratory problems. During these studied, molds were isolation and identification based on Conventional method (Direct microscopy by using 10% KOH, and fungal culture was done on Sabouraud Dextrose agar supplemented with chloramphenicol and on Czapek-Dox agar incubated at 37°C and examined for 3-7 days then macroscopic, microscopic examination of the colony by(lactophenol cotton blue stain )and molecular methods by using Polymerase chain reaction (PCR)technique for identification. The 10% KOH examination was positive for 35 cases, while laboratory culturing was positive for 53 cases. Aspergillus sp were isolated from 44(83%) patients; A. fumigatus was isolated in 23 (42. 4%) patients while A. flavus, A. niger, and A. terreus were isolated from 11 (20. 08%), (13. 2%) and 3 (5. 7%) patients respectively, also isolated Penicillium spp. at percentage 1(1. 9%). In this study. The ages of participants ranged from 10-70years with a mean age of 34years, the males were more susceptible to fungal infection, were recorded 35/53 (66. 3), compared to females were 18/53 (33. 96). The infection of fungi was more prevalent in ages 30-40recorded 26(53. 06%) followed by ages 40-50, 13(26. 5), while the lowest infection recorded in the age group 10- 20 years was 2(2. 04%). DNA isolated from twenty-three A. fumigatus isolates was used as a template, and the specific of oligonucleotide primer sequences were used in conventional PCR to detect the presence of internal transcribed spacer region ( ITS) region of the rRNA gene for Aspergillus fumigates. The results of the PCR amplification of the rRNA gene showed that this gene was present in 19 samples out 23 positive samples which isolation with a PCR product size of approximated 385 bp, while 4 samples out 23 positive samples showed negative results for the presence of this gene as indicated by the absence of the PCR products in their relevant lanes. Statistical analysis revealed that the PCR to have a sensitivity of 95. 1% in the detection of Aspergillus fumigatus in Aspergillosis cases. Polymerase chain reaction (PCR) is a rapid, specific, and sensitive method to detect Aspergillus fumigatus in aspergillosis cases of humans.

Regulation mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice

Objective: To explore the regulatory mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice. Methods: E2F1 gene knockout heterozygotes C57BL/6 mice and wild-type C57BL/6 mice were introduced and self-reproduced. Two weeks after birth, E2F1 gene knockout homozygotes mice and wild-type mice were identified by polymerase chain reaction (PCR). Twelve identified 6-8 weeks old male E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were selected respectively according to the random number table and set as E2F1 gene knockout group and wild-type group. A full-thickness skin defect wound was made on the back of each mouse. On post injury day (PID) 2 and 7, 6 mice in each group were selected according to the random number table and sacrificed, and the wound tissue was excised. The expression of CD68 and CD206 double positive M2 macrophages was observed by immunofluorescence method, and the percentage of CD206 positive cells was calculated. The protein expression of CD206 was detected by Western blotting. The mRNA expression of arginase 1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-PCR). Wound tissue specimens of the two groups on PID 7 were obtained, and the protein and mRNA expressions of peroxisome proliferator-activated receptor gamma (PPAR-γ) were detected by Western blotting and real-time fluorescent quantitative RT-PCR respectively. The above-mentioned experiments were repeated four times. Three specimens of wound tissue of mice in wild-type group on PID 7 were obtained to detect the relationship between E2F1 and PPAR-γ by co-immunoprecipitation and Western blotting, and this experiment was repeated two times. Data were processed with unpaired t test. Results: The size of PCR products of E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were 227 and 172 bp respectively, which were the same as those of the designed DNA fragments. On PID 2 and 7, the number of CD68 and CD206 double positive M2 macrophages in the wound tissue of mice in E2F1 gene knockout group was more than that of wild-type group, and the percentages of CD206 positive cells in the wound tissue of mice in E2F1 gene knockout group were (0.234±0.032)% and (0.584±0.023)% respectively, which were significantly higher than (0.129±0.017)% and (0.282±0.071)% of wild-type group (t=3.29, 3.54, P<0.05). On PID 2 and 7, the protein expression of CD206 in the wound tissue of mice in E2F1 gene knockout group were 1.00±0.23 and 1.63±0.26 respectively, which were significantly higher than 0.43±0.06 and 0.97±0.08 of wild-type group (t=2.41, 2.45, P<0.05). On PID 2 and 7, the mRNA expressions of arginase 1 in the wound tissue of mice in E2F1 gene knockout group were 0.482±0.105 and 0.195±0.031 respectively, which were significantly higher than 0.163±0.026 and 0.108±0.017 of wild-type group (t=3.04, 2.86, P<0.05). On PID 7, the protein and mRNA expressions of PPAR-γ in the wound tissue of mice in E2F1 gene knockout group were 0.61±0.12 and 0.51±0.13 respectively, which were significantly higher than 0.20±0.04 and 0.20±0.04 of wild-type group (t=3.36, 2.86, P<0.05). On PID 7, detection of the wound tissue of mice in wild-type group showed that PPAR-γ had unidirectional effect on E2F1. Conclusions: E2F1 transcription factor affects the polarization of M2 macrophages by inhibiting the expression of PPAR-γ, thereby inhibiting the healing process of full-thickness skin defect wounds in mice.

Detection of Aspergillus fumigatus by Polymerase Chain Reaction(PCR)

Aspergillosis refers to fungi infections of the respiratory tract caused by Aspergillus species especially Aspergillus fumigatus.Infection of A. fumigatus were increased in last few years , due to either resistances to antibiotics or to influence of other factors such as other fungal infections.The aims of the present study were to review the impact of Aspergillus fumigatus in Aspergillosis cases, and study the role of Singleplex PCR for amplification of ITS1,ITS4 of rRNA gene in the detection of fungal isolate . In this study One hundred sputum samples were collected from patients admitted to the specialize chest and respiratory diseases center / Baghdad who were suffering from respiratory problems. During these studied, molds were isolation and identification based on Conventional method (Direct microscopy by using 10% KOH, and fungal culture was done on Sabouraud Dextrose agar supplemented with chloramphenicol and on Czapek-Dox agar incubated at 37°C and examined for 3-7 days then macroscopic, microscopic examination of the colony by(lactophenol cotton blue stain )and molecular methods by using Polymerase chain reaction (PCR)technique foridentification. The 10% KOH examination was positive for 35 cases while laboratory culturing was positive for 53 cases. Aspergillus sp were isolated from 44(83%) patients; A. fumigatus was isolated in 23 (42.4%) patients while A. flavus, A. niger, and A. terreus were isolated from 11 (20.08%), (13.2%) and 3 (5.7%) patients respectively, also isolated Penicillium spp. at percentage 1(1.9%).. In this study.The ages of participants ranged from 10-70years with a mean age of 34years, the males were more susceptible for fungal infection were recorded 35/53 (66.3), compared to females were 18/53 (33.96).The infection of fungi were more prevalent in ages 30-40recorded 26(53.06%) followed by ages 40-50 ,13(26.5), while the lowest infection recorded in age group 10- 20 years was 2(2.04%). DNA isolated from twenty three A.fumigatus isolates was used as template and the specific of oligonucleotide primer sequences were used in conventional PCR to detect the presence of internal transcribed spacer region ( ITS) region of the rRNA gene for Aspergillus fumigates. The results of the PCR amplification of the rRNA gene showed that, this gene was present in 19 samples out 23 positive samples which isolation with a PCR product size of approximated 385 bp, while 4 samples out 23 positive samples showed negative results for the presence of this gene as indicated by the absence of the PCR products in their relevant lanes.Statistical analysis revealed that the PCR to have a sensitivity of 95.1 % in the detection of Aspergillus fumigatus in Aspergillosis cases.Polymerase chain reaction (PCR) is a rapid, specific and sensitive method to detect Aspergillus fumigatus in aspergillosis cases of human.

PCR-Based test for identification of meat of ovine, caprine and bovine origin

Polymerase chain reaction (PCR) test has been developed for qualitative differentiation of meat of ovine (Ovis aries), caprine (Capra hircus) and bovine (Bos indicus) origin. Novel primers for detection of bovine origin meat were designed to target mitochondrial D-loop region. Different sizes of the species-specific PCR products were separated by agarose gel electrophoresis for identification of all species. Sensitivity of the bovine specific primers was tested with two-fold serial dilutions of genomic DNA. Amplification was achieved with 20 ng of genomic DNA. The specificity of the developed test was cross checked with the DNA of sheep, goat and buffalo, where amplification was not observed. This test is handy for accurate identification of meat of three animal species. This test will help in preventing the mislabelling and fraudulent substitution of meat and meat product for economic gain.

POLYMORPHISM OF METHYLENETETRAHYDROFOLATE REDUCTASE (A1298C) AS A RISK FACTOR FOR OSTEOPOROSIS IN POST-MENOPAUSAL INDONESIAN WOMEN

Objective: The study aimed to assess the association between the A1298C polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene and risk to osteoporosis in post-menopausal Indonesian women.Methods: After ethical approval, calcaneus bone mineral density (BMD) (T-scoring) was assessed from 194 consenting post-menopausal Indonesian women by dual-energy X-ray absorptiometry, and DNA of the participants was isolated from peripheral blood samples. To determine the status of A1298C polymorphism of MTHFR, isolated DNA was polymerase chain reaction (PCR) amplified in 35 cycles, to result in PCR product sizes of 302 bp (1298A) and 275 bp (1298C). For fragment detection, the PCR product was subjected to electrophoresis on agarose gel. The results were statistically assessed for genotype and allotype comparison according to the T-score grouping by Chi-square analysis, assuming statistical significance at p&lt;0.05.Results: The results show no significant association between the T-score (BMD) grouping and genotype (or allotype) of the tested polymorphism of MTHFR. The observed genotype distribution of the tested MTHFR polymorphism (A1298C) differs clearly from those previously reported, with AC as the dominant genotype in the Indonesian sample population of the present work.Conclusion: The MTHFR (A1298C) polymorphism is relatively infrequent in the Indonesian female population, and no significant association was observed between this polymorphism, bone mineral density, and osteoporotic status.

Development of High Quality EST-SSR Markers Without Stutter Bands in Peach and Their Application in Cultivar Discrimination and Hybrid Authentication

Peach (Prunus persica) is an important fruit crop worldwide with several thousand cultivars. Cultivar discrimination and hybrid authentication are often required in peach breeding and can be achieved by applying various molecular markers including simple sequence repeat (SSR). In this study a total of 2146 expressed sequence tag (EST)–SSR loci were detected with the 10,737 EST sequences retrieved from the NCBI. A total of 49 EST-SSR markers, including 24 simple ones with a motif comprising of tri-, tetra-, penta-, hexanucleotides, and 25 compound ones, were selected and then primers were designed. Following conventional polymerase chain reaction (PCR) specificity control and sequence authentication, as well as fluorescence-based PCR product size and stutter band evaluation, 37 EST-SSR markers with correct amplification and without stutter band interference were validated. Among them, 14 were polymorphic in 18 closely related peach accessions, with polymorphism information content (PIC) ranging from 0.0994 to 0.3750. The 18 peach accessions can be distinguished using nine polymorphic markers, with the exception of ‘Shangshandayulu’ and ‘Xipu 1’, both being bud sports from ‘Yulu’. The clustering of the accessions as well as the fingerprint profiles supported the authentication of the hybrids. These EST-SSR markers are useful for peach breeding research.

Authentication of sheep (Ovis aries) and goat (Capra hircus) meat species using species-specific polymerase chain reaction

Small ruminants (sheep and goat) immensely contribute to the Indian meat food basket; yet regional preference for either sheep or goat meat varies. Owing to the differences in the market prices, these two closely resembling species meats are often misrepresented interchangeably. Prevention of such fraudulent practices requires authentic and sensitive speciation tools. A novel mitochondrial D-loop based species-specific polymerase chain reaction (PCR) technique was developed for the precise detection of origin of sheep and goat meats. Species-specific primers (SSP-G and SSP-S) developed for PCR in this study amplify 229 and 425 base pairs products specific to goat and sheep species. An additional herteroplasmic PCR product of size 350 bp was evident only in sheep. Primers designed in this study were checked for the absence of cross-species amplification in closely related animal species and was successfully applied for the authentication of a forensic case. Our results indicated prerogative applications of the novel species-specific PCR for the authentic identification of origin of sheep and goat species thereby aiding in the prevention of the menace of misrepresentation of meats.

MICA*A4 protects against ulcerative colitis, whereas MICA*A5.1 is associated with abscess formation and age of onset.

Ulcerative colitis (UC) is one of the two major forms of inflammatory bowel disease, the aetiology of which remains unknown. Several studies have demonstrated the genetic basis of disease, identifying more than 130 susceptibility loci. The major histocompatibility complex class I chain-related gene A (MICA) is a useful candidate to be involved in UC pathogenesis, because it could be important in recognizing the integrity of the epithelial cell and its response to stress. The aim of this study was to analyse the relationship between polymorphisms in the transmembrane domain of MICA and susceptibility to develop UC. A total of 340 patients with UC and 636 healthy controls were genotyped for MICA transmembrane polymorphism using a polymerase chain reaction (PCR) combined with fluorescent technology. Different MICA alleles were determined depending on the PCR product size. The allele MICA*A4 was less frequent in patients than in controls (P = 0·003; OR = 0·643), and this protective role is higher when it forms haplotype with B*27 (P = 0·002; OR = 0·294). The haplotype HLA-B*52/MICA*A6 was also associated with UC [P = 0·001; odds ratio (OR) = 2·914]. No other alleles, genotypes or haplotypes were related with UC risk. Moreover, MICA*A5.1 is associated independently with abscesses (P = 0·002; OR = 3·096) and its frequency is lower in patients diagnosed between ages 17 and 40 years (P = 0·007; OR = 0·633), meaning an extreme age on onset. No association with location, extra-intestinal manifestations or need for surgery was found.

Clonal relationships in recurrent B-cell lymphomas.

Immunoglobulin (Ig) gene rearrangements remain largely unmodified during the clonal expansion of neoplastic cells. We investigated the clonal relationships between lymphoma components at diagnosis and at relapse by analyzing Ig gene rearrangements. A BIOMED-2 multiplex polymerase chain reaction (PCR) assay was performed in 27 patients using formalin-fixed paraffin embedded tissues, with subsequent cloning and sequencing of the amplified Ig genes in 17 patients. All 27 cases of primary and corresponding relapsed tumors showed monoclonal rearrangements of the Ig genes by BIOMED-2 PCR. Whereas IgVH or IgVK fragment lengths were identical in 8/27 pairs (30%), fragment lengths differed in 19/27 pairs (70%). In 17 cases analyzed by sequencing, an identical VDJ gene rearrangement was confirmed in 4/4 pairs (100%) with the same fragment lengths and in 10/13 pairs (77%) with different fragment lengths. Four of 17 primary lymphomas had multiple VDJ rearrangements, and three of them showed an unrelated relapse. Unrelated relapse was observed in 1/8 mantle cell lymphomas, 1/5 diffuse large B-cell lymphomas, and a large B cell lymphoma developed in a patient with a small lymphocytic lymphoma. Unrelated relapses developed after a longer disease-free interval and tended to show poorer outcome compared with related relapse. In summary, relapse of a lymphoma from an unrelated clone is uncommon, but can occur in B-cell lymphomas. Clonal relationships should be determined by sequencing of the Ig genes, and not just by comparing the PCR product size.

Multiplex PCR for simultaneous identification of turkey, ostrich, chicken, and duck

To discriminate economically motivated adulteration in poultry meats and products, species-specific oligonucleotide primer sets for multiplex polymerase chain reaction (PCR) of four poultry meats were newly designed. Sequences for species-specific primers were generated based on the nucleotide variation in mitochondria cytochrome b gene through sequence comparisons. The sizes of species-specific PCR products for turkey, ostrich, chicken, and duck were 217, 330, 516, and 820 bp, respectively. By adjusting PCR conditions and the concentrations of primers, a simultaneous discrimination of four poultry meats was successfully performed. The limits of detection of both single and multiplex PCR for turkey were 0.05 and 0.005 ng for other species, respectively. The developed multiplex PCR method was successfully applied to identify the target species in 34 of poultry meat products and thus was considered to be a useful tool for monitoring poultry meat products.

Molecular approach for identification of fungi from soils of Cameron Highlands

Seven cultures of fungi were isolated from the soil of Cameroon Highlands. In this research, molecular approach was used for the identification of these fungi. The genomic DNA of the fungi species was extracted and the target sequences within the DNA were amplified using polymerase chain reaction (PCR) with ITS 1 (forward) and ITS 4 (reverse) as the primers which amplified the Internal Transcribe Spacer (ITS) of the fungi. The size of PCR product was 600 base pairs. The PCR products were purified and the DNA purity obtained for culture 1, 2, 3 and 4 were 1.74, 1.92, 1.83 and 1.77, respectively. Based on the sequences of the PCR products, three species were successfully identified as Aspergillus flavus, Aspergillus niger and Fusarium oxysporum.Â

Molecular characterization of African swine fever virus from domestic pigs in northern Tanzania during an outbreak in 2013.

African swine fever (ASF) is an acute, highly contagious and deadly viral hemorrhagic fever of domestic pigs caused by African swine fever virus (ASFV), a double-stranded DNA virus of the family Asfarviridae. In this study, molecular diagnosis and characterization of outbreak ASFV in northern Tanzania, was performed on spleen, lymph node, kidney, and heart samples collected in June and July 2013 from domestic pigs that died during a hemorrhagic disease outbreak. Confirmatory diagnosis of ASF was performed using polymerase chain reaction (PCR) by partial amplification of B646L gene of ASFV encoding the major capsid protein p72 using PPA1/PPA2 primers. PCR using PPA1/PPA2 primers produced an expected PCR product size, confirming ASF outbreak in northern Tanzania. In addition, nucleotide amplification and sequencing, and phylogenetic reconstruction of the variable 3′-end of the B646L gene and complete E183L gene encoding the inner envelope transmembrane protein p54 showed that the 2013 outbreak ASFV from northern Tanzania were 100 % identical and clustered into ASFV B646L (p72) and E183L (p54) genotype X. Furthermore, the tetrameric amino acid repeats within the central variable region (CVR) of the B602L gene coding for the J9L protein had the signature BNBA(BN)5NA with a single novel tetramer NVDI (repeat code N). The results of the present study confirm an ASF outbreak in northern Tanzania in the year 2013 and show that the present outbreak ASFV is closely related to other ASFV from ticks, warthogs, and domestic pigs previously reported from Tanzania.

Diagnosis of Mycobacterium bovis in Captive Sloth Bears (Melursus ursinus) by Polymerase Chain reaction

Diagnosis of mycobacterial infection is paramount important from the public health perspective since treatment and control measures are very significant, particularly in captive animals. In this diagnostic study of Mycobacterium bovis infection in sloth bears (Melursus ursinus), polymerase chain reaction (PCR) had been used with the primer sequence of pncA-8 (5′-GGTTGGGTGGCCGCCGGTCAG-3′) and pncA-11 (5′-GCTTTGCGGCGAGCGCTCCA-3′) that were specific for M. bovis pncA gene. Forty-two fresh faecal samples were collected randomly from the apparently healthy sloth bears maintained in captive conditions. The DNA extraction procedure was done as per the manufacturer’s protocol and further subjected to amplification. The amplification profile includes respectively: initial heating of the samples for 5 min at 94 °C, annealing at 55 °C for 1 min, primer extension at 72 °C for 1 min and final elongation step for 10 min at 72 °C. Out of 57 samples, 5 samples were yielded on expected amplified PCR product size of 744 bp when electrophoresed in 1.5 % agarose gel. A positive control of M. bovis DNA procured from Tuberculosis Research Centre and a negative control from a healthy bovine sample were used. These results demonstrated that PCR test will increase the effectiveness of laboratory diagnosis to detect and identifying the M. bovis in captive wild animals.

Genomic Diversity and Virulence Genes among Clinical Isolates of Pseudomonas aeruginosa

Typing of nosocomial pathogens is necessary to determine the source of an outbreak. The aim was to determine the genomic variability among Pseudomonas aeruginosa (P. aeruginosa) by random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) methods. Fifty P. aeruginosa isolates were obtained from the hospitals. The source of these isolates were burn wound and urinary tract infections. After detection of P. aeruginosa by biochemical methods, chromosomal deoxyribonucleic acid (DNA) was extracted by a DNA extraction kit. ERIC-PCR and RAPD- PCR was done by standard methods. The polymerase chain reaction (PCR) products were run and visualized in 1.5% agarose gels stained with ethidium bromide. Fifty P. aeruginosa isolates were analyzed by ERIC-PCR and RAPD-PCR methods. Multiple PCR fragment sizes generated by two PCR methods and PCR product size were between 200-3500 bp, and 10 and 7 different PCR patterns were detected by ERIC-PCR and RAPD-PCR, respectively. Eleven isolates were not detected by ERIC-PCR method. Fifteen isolates were typed to a single genotype by the RAPD-PCR method. We suggested that ERIC and RAPD PCR are equally suitable, inexpensive, fast, reproducible, and discriminatory as rapid DNA typing tools for effective epidemiological surveillance of P. aeruginosa isolates. Our results suggest that these DNA typing tools could be used in routine epidemiological surveillance, outbreak surveillance, and in the identification of the source of transmission of P. aeruginosa.

Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma.

To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers. The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis. CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC. CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.