Sequencing Of Polymerase Chain Reaction Products Research Articles

Towards pharmacogenomics-guided tuberculosis (TB) therapy: N-acetyltransferase-2 genotypes among TB-infected Kenyans of mixed ethnicity

BackgroundThough persons of African descent have one of the widest genetic variability, genetic polymorphisms of drug-metabolising enzymes such as N-Acetyltransferase-2 (NAT2) are understudied. This study aimed to identify prevalent NAT2 single nucleotide polymorphisms (SNPs) and infer their potential effects on enzyme function among Kenyan volunteers with tuberculosis (TB) infection. Genotypic distribution at each SNP and non-random association of alleles were evaluated by testing for Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD).MethodsWe isolated genomic DNA from cryopreserved Peripheral Blood Mononuclear Cells of 79 volunteers. We amplified the protein-coding region of the NAT2 gene by polymerase chain reaction (PCR) and sequenced PCR products using the Sanger sequencing method. Sequencing reads were mapped and aligned to the NAT2 reference using the Geneious software (Auckland, New Zealand). Statistical analyses were performed using RStudio version 4.3.2 (2023.09.1 + 494).ResultsThe most frequent haplotype was the wild type NAT2*4 (37%). Five genetic variants: 282C > T (NAT2*13), 341 T > C (NAT2*5), 803A > G (NAT2*12), 590G > A (NAT2*6) and 481C > T (NAT2*11) were observed with allele frequencies of 29%, 18%, 6%, 6%, and 4% respectively. According to the bimodal distribution of acetylation activity, the predicted phenotype was 76% rapid (mainly consisting of the wildtype NAT2*4 and the NAT2*13A variant). A higher proportion of rapid acetylators were female, 72% vs 28% male (p = 0.022, odds ratio [OR] 3.48, 95% confidence interval [CI] 1.21 to 10.48). All variants were in HWE. NAT2 341 T > C was in strong complete LD with the 590G > A variant (D′ = 1.0, r2 = − 0.39) but not complete LD with the 282C > T variant (D′ = 0.94, r2 = − 0.54).ConclusionThe rapid acetylation haplotypes predominated. Despite the LD observed, none of the SNPs could be termed tag SNP. This study adds to the genetic characterisation data of African populations at NAT2, which may be useful for developing relevant pharmacogenomic tools for TB therapy. To support optimised, pharmacogenomics-guided TB therapy, we recommend genotype-phenotype studies, including studies designed to explore gender-associated differences.

Microsporidial keratoconjunctivitis - first outbreak in Japan.

Most cases of microsporidial keratoconjunctivitis are found in the Southern hemisphere. Our purpose was to investigate the first outbreak of microsporidial keratoconjunctivitis in Japan among healthy, immunocompetent soccer players from the same team during a 1-month period. This study is an observational case series. The medical records were analyzed for five cases with microsporidial keratoconjunctivitis who presented within September 2022. All five cases were males between 28 and 36 years old. These previously healthy individuals belonged to the same football team. Their eyes were considered susceptible to contaminated water or dirt from the turf at game and practice sites. All cases involved unilateral conjunctivitis, with scattered round white lesions that showed positive fluorescein staining in the corneal epithelium. All cases experienced diminution of vision in the affected eye. In three cases, direct smears showed spores of approximately 2-3μm in diameter. Polymerase chain reaction (PCR) analysis of corneal scrapes revealed partial amplification of microsporidial 18S ribosomal RNA gene in four cases. Sequences of PCR products from all four cases showed 100% identity with strains of Vittaforma corneae previously reported from an outbreak in Singapore. All cases were treated with topical therapy, including voriconazole, fluorometholone, and levofloxacin. Four eyes underwent corneal scraping. After treatment, all eyes healed without residual opacities. Only a few sporadic case reports of this disease have previously been reported in Japan. We detected V. corneae in our case series, representing what appears to be the first outbreak of microsporidial keratoconjunctivitis in Japan. Exposure to contaminated water or soil, in addition to inadequate sanitary facilities, represents a potential source of infection. Further investigations to clarify the characteristics of microsporidia seem warranted.

Molecular evidence of Rickettsia canadensis in ticks, Hebei, China

China was affected severely by tick-borne rickettsiosis, and more than 10 Rickettsia species pathogenic to humans have been identified. In recent years, several Rickettsia members, with unknown pathogenicity, firstly identified abroad have been found in China. In this study, parasitic and questing ticks were recovered from two sampling sites in Hebei, China. Specific primers targeting outer membrane protein B (ompB) gene were designed to test the presence of Rickettsia canadensis by nested Polymerase Chain Reaction (PCR). As a result, a total of 428 ticks, including 232 ticks (including 230 Haemaphysalis longicornis and two H. japonica) from Laiyuan County and 196 (H. longicornis) from Luanping County, were collected. Sequencing of PCR products with the expected size and subsequently BLAST showed that 38H. longicornis ticks tested positive for R. canadensis, with an overall positive rate of 8.8%. In addition, 800-bp ompB gene and nearly complete citrate synthase (gltA) gene were recovered from six randomly selected positive samples to better understand their genetic characteristics. Nucleotide identity and phylogenetic analyses showed that R. canadensis presented geographical clustering with evidence that variants identified in the current study presented closer genetic relationship with others identified in Asian than those found in North America. In addition, epidemiological data suggested that H. longicornis may be the competent vector, and more attention should be paid to R. canadensis due to its zoonotic potential. In sum, R. canadensis was confirmed to be present in Hebei Province, China, and its surveillance in ticks should be strengthened due to potential pathogenicity, higher positive rate in ticks and wide distribution of possible vector tick species.

BRAF V600E Mutation of Non-Small Cell Lung Cancer in Korean Patients.

Background and Objectives: BRAF mutational status in resected non-small cell lung cancer (NSCLC) in the Korean population is poorly understood. We explored BRAF (particularly BRAF V600E) mutational status among Korean patients with NSCLC. Materials and Methods: This study included 378 patients with resected primary NSCLC who were enrolled from January 2015 to December 2017. The authors obtained formalin-fixed paraffin-embedded (FFPE) tissue blocks and performed peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR) for detecting BRAF V600, real-time PCR for detecting BRAF V600E, and immunohistochemical analyses using the mutation-specific Ventana VE1 monoclonal antibody. For positive cases in any methods mentioned above, direct Sanger sequencing was additionally performed. Results: The PNA-clamping method revealed the BRAF V600 mutation in 5 (1.3%) of the 378 patients. Among these five patients, real-time PCR, direct Sanger sequencing detected BRAF V600E mutations in three (0.8%) patients. Thus, two cases showed differences in their PNA-clamping and the others. Direct Sanger sequencing of PNA-clamping PCR product was performed for two cases showing negative results on direct Sanger sequencing; both contained BRAF mutations other than V600E. All patients harboring BRAF mutations had adenocarcinomas, and all patients with V600E mutation exhibited minor micropapillary components. Conclusions: Despite the low incidence of the BRAF mutation among Korean patients with NSCLC, lung adenocarcinoma patients with micropapillary components should be prioritized in terms of BRAF mutation testing. Immunohistochemical staining using Ventana VE1 antibody may serve as a screening examination for BRAF V600E.

Diversity of Chlamydiales detected in pet birds privately kept in individual homes in Japan.

Chlamydia-related bacteria of the Chlamydiales order have recently been described as emerging pathogens that cause pneumonia and abortion in animals and humans. We investigated the presence of Chlamydiales using real-time polymerase chain reaction (PCR) by targeting the 16S rRNA gene of a broad range of Chlamydiales in 827 fecal samples from pet birds kept in individual homes in Japan. Of the 827 samples, 493 (59.6%) tested positive for the Chlamydiales 16S rRNA gene in the real-time PCR assay. We determined the nucleic acid sequences of PCR products from 17 Chlamydiales strains. A homology search and phylogenetic analysis using these sequences confirmed that the detected Chlamydiales included C. pecorum and a broad range of Chlamydia-related bacteria. To the best of our knowledge, this is the first study to detect a wide range of Chlamydia-related bacteria in birds.

Sulfate-Reducing Bacteria in Patients Undergoing Fixed Orthodontic Treatment

ObjectivesThe oral microbiological environment may be implicated in the corrosion of orthodontic metals. This study aimed to examine the prevalence of sulfate-reducing bacteria (SRB) in orthodontic patients undergoing fixed appliance treatment. MethodsSixty-nine orthodontic and 69 healthy non-orthodontic participants were enrolled in the study. Supragingival and subgingivaloral biofilm were collected and tested for the presence of SRB. The DNA extraction, polymerase chain reaction (PCR), and 16sRNA Sanger sequencing method was performed from the SRB-positive samples. The sequenced PCR products were analysed and compared with databases to identify the bacterial genus. ResultsAmongst 69 orthodontic patients, characteristic black precipitates developed in 14, indicating the presence of iron sulfides which demonstrates the likelihood of SRB. Alternatively, 2 out of 69 showed the presence of SRB in healthy non-orthodontic participants (controls). Desulfovibrio spp was confirmed by analyses of 16sRNA sequencing, which revealed that the SRB prevalence was 20% in the examined participants with orthodontic appliances. ConclusionsThe prevalence of SRB was found to be significantly higher amongst orthodontic patients compared to non-orthodontic participants. Presence of stainless steel in the oral environment may have facilitated the colonisation of SRB.

Epidemiology, clinical features, and molecular detection of orf virus in Haryana (India) and its adjoining areas.

Orf is an acute, highly contagious, and economically important viral disease of small ruminants. In this study, six orf suspected outbreaks among goats and sheep were investigated from Haryana state and adjoining areas of Rajasthan state during the year 2021. The disease was diagnosed on the basis of clinical signs and molecular identification. The causative agent of the disease, orf virus (ORFV), was confirmed using polymerase chain reaction (PCR) targeting immunodominant envelope antigen (B2L) gene and confirmed by sequencing. The morbidity in goats ranged from 8.75 to 100%, whereas in sheep, it ranged from 0 to 8%. The higher mortality was observed among flocks with mixed infections of orf and peste des petits (PPR) or orf and haemonchosis as compared to other outbreaks. The phylogenetic analysis of sequenced PCR products clustered the current study strains in the same clad with Indian as well as strains from other countries with nucleotide identity more than 99%, signifying a close genetic relationship. The study highlighted the circulation of strains of a single cluster among sheep and goats in Haryana and adjoining areas. Prompt diagnosis of the disease is highly important for facilitating the implementation of control measures to minimize the losses suffered by small and marginal farmers in this region. Further detailed studies are required to delineate the molecular details of ORFV for better understanding the dynamics and molecular epidemiology of strains circulating in the country and for designing the effective vaccines against the disease which are currently lacking in the country.

Candidate Genes in Testing Strategies for Linkage Analysis and Bioinformatic Sorting of Whole Genome Sequencing Data in Three Small Japanese Families with Idiopathic Superior Oblique Muscle Palsy.

Idiopathic superior oblique muscle palsy is a major type of paralytic, non-comitant strabismus and presents vertical and cyclo-torsional deviation of one eye against the other eye, with a large vertical fusion range and abnormal head posture such as head tilt. Genetic background is considered to play a role in its development, as patients with idiopathic superior oblique muscle palsy have varying degrees of muscle hypoplasia and, rarely, the complete absence of the muscle, that is, aplasia. In this study, whole genome sequencing was performed, and single nucleotide variations and short insertions/deletions (SNVs/InDels) were annotated in two patients each in three small families (six patients in total) with idiopathic superior oblique muscle palsy, in addition to three normal individuals in one family. At first, linkage analysis was carried out in the three families and SNVs/InDels in chromosomal loci with negative LOD scores were excluded. Next, SNVs/InDels shared by the six patients, but not by the three normal individuals, were chosen. SNVs/InDels were further narrowed down by choosing low-frequency (<1%) or non-registered SNVs/InDels in four databases for the Japanese population, and then by choosing SNVs/InDels with functional influence, leading to one candidate gene, SSTR5-AS1 in chromosome 16. The six patients were heterozygous for 13-nucleotide deletion in SSTR5-AS1, except for one homozygous patient, while the three normal individuals were wild type. Targeted polymerase chain reaction (PCR) and direct sequencing of PCR products confirmed the 13-nucleotide deletion in SSTR5-AS1. In the face of newly-registered SSTR5-AS1 13-nucleotide deletion at a higher frequency in a latest released database for the Japanese population, the skipping of low-frequency and non-registration sorting still resulted in only 13 candidate genes including SSTR5-AS1 as common variants. The skipping of linkage analysis also led to the same set of 13 candidate genes. Different testing strategies that consisted of linkage analysis and simple unintentional bioinformatics could reach candidate genes in three small families with idiopathic superior oblique muscle palsy.

USING sma-its2 PRIMER FOR ITS2 (Internal Transcribed Spacer-2)-BASED MOLECULAR CHARACTERIZATION OF Anopheles minimus FROM KULONPROGO, YOGYAKARTA - INDONESIA

Malaria is a public health threat caused by the Plasmodium infection transmitted by the Anopheles mosquito. Malaria vector control is highly dependent on the ability to determine mosquito species' vectorial and bionomic capacity. Species identification based on morphological characteristics as well as DNA-barcoding approaches is a very important step in determining vectorial capacity. Our research group has redesigned a new ITS2 primer, namely, sma-its2, which is specifically able to identify the Anopheles (An.) mosquito vector but cannot identify other mosquito vectors. This study wanted to test this primer's specificity further for identification of other Anopheles mosquitoes. We used An. minimus collected from Kulonprogo, Yogyakarta – Indonesia. The methods used in this research are as follows: landing collection, morphological identification, isolation of genomic DNA, PCR (Polymerase Chain Reaction), PCR product purification, sequencing, and data analysis. An. vagus from Bangsring, Banyuwangi – Indonesia, which had previously been identified using the same primary, was used as a positive control. The results of the morphological analysis showed that both species were in accordance with the vector identification key used in this study. The molecular analysis showed that the sma-its2 primer could amplify the ITS2 sequence of An. vagus and An. minimus, producing 650 – 700 bp. However, further analysis of the ITS2 sequences of both species, resulted in the same species, namely An. vagus, with a different accession number in GenBank. This showed that the sma-its2 primar can be used to identify An. vagus but cannot be used to identify An. minimus. Analysis of the primer position in the ITS2 sequences showed the presence of 3 nucleotides in the forward sma-its2 primer that was not recognized by the An. minimus sequences and thus, hinder the successful identification of these species.

Genetic Analysis of Alpha-Thalassemia Mutations in Thi-Gar Province, Iraq.

The prevalence of alpha-thalassemia as a major health problem in the south of Iraq has highlighted the necessity of investigations and screening of patients with thalassemia. The present study aimed to characterize the spectrum of alpha-globin gene mutations in patients who were followed up in a genetic diseases center in Thi-Qar province. A total of 30 subjects were collected from thalassemia patients and 15 cases as the control group. Polymerase chain reaction (PCR) and direct sequencing were performed for functionally regions of the gene (exon 1 and exon 2). The fragment size amplified was 442 bp in the Exon 1 region and 324 bp in the Exon 2 region of α-globin. The molecular analysis of the sequence of PCR products revealed that 13 point mutation within the α-thalassemia gene included deletion and substitution mutation, while the rest of the mutations were in the intron site of the gene. These results indicated that mutations may constitute a risk of developing hemophilia B disease. Molecular mechanisms in the expression of globin genes are used to help manage patients with thalassemia.

Molecular detection of Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania

A growing number of Campylobacter species other than C. jejuni and C. coli have been considered as emerging human and animal pathogens but their contribution to human gastroenteritis is poorly documented. This study aimed at detecting Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania using molecular techniques without culture. Seventy (70) faecal samples were collected from five diarrheic and 65 non-diarrheic human patients attending Kilosa District Hospital in Tanzania from July to October 2019. During the same period, 30 faecal samples were also collected from healthy cattle in the same district. Genus and species identification of Campylobacter was conducted on the samples using molecular techniques [the polymerase chain reaction (PCR) and 16S rRNA sequencing]. Phylogenetic analysis was carried out by comparison of the 16S rRNA gene sequences to reference strains by the Neighbor-Joining method in MEGA X. Campylobacter species detection rate by PCR was 65.7% (46/70) and 20% (6/30) in humans and cattle, respectively. There were five human diarrheic cases, four of which were positive for Campylobacter and of these, two were children ≤15 years of age. In humans, 16S rRNA sequencing revealed that C. concisus was the most predominant species occurring at a frequency of 37.8% (14/37), followed by uncultured Campylobacter spp. 24.3% (9/37) and C. hominis 21.6% (8/37). The least represented species were C. jejuni and C. lanienae, all occurring at 2.7% (1/37). In cattle, five (100%) sequenced PCR products matched with C. lanienae. Phylogenetic analysis revealed that with the exception of C. lanienae, 16S rRNA sequences of Campylobacter species were closely related to the reference strains used (Percent identity: 90.51-96.56%). Based on our findings, we recommend that molecular techniques, mainly PCR be adopted for the direct detection of Campylobacter species during laboratory screening and surveillance studies.

Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas

Vibrio bacteria are one of the greatest threats to aquaculture, with Vibrio-associated mass mortality events occurring in all life stages worldwide. Vibrio aestuarianus is particularly problematic in Pacific oyster Crassostrea gigas cultures. A diagnostic method used to detect V. aestuarianus is quantitative (q)PCR and a confirmatory method is fluorescent in situ hybridization (FISH). These methods require specialized equipment and incur high costs. The objective of this study was to develop a novel generic (relating to a genus) PCR and DIG-labeled in situ hybridization (ISH) for the detection and confirmation of Vibrio. A PCR that detects all Vibrio spp. is particularly useful for blanket screening of hosts and environmental samples, while ISH facilitates the visual localization of bacteria. Both diagnostic methods were developed to be used synchronously with other methods that are specific to certain Vibrio spp. Primers (VibF3/VibR3) were designed to amplify a 286 bp product from the 16S ribosomal RNA gene common to all Vibrio spp. and to form the ISH probe. Confirmatory positive and negative controls were used in both methods. Results were compared with a V. aestuarianus specific qPCR protocol. Direct Sanger sequencing of PCR products, Blastn analysis and Clustal Omega alignments were used to confirm Vibrio sp. detection and assess similarity. Pathologies observed in C. gigas tissues were similar to those described in other studies. A positive ISH signal was observed in qPCR and PCR positive oysters and no signal was detected in uninfected individuals. These techniques will increase the diagnostic arsenal against this globally significant genus of bacteria.

Detection of Genetic Polymorphism of HER2 Gene in HER2 Positive Breast Cancer Women in Iraq

The human epidermal growth factor receptor-2 (HER2) gene plays a critical role in breast cancer development and progression. HER2 overexpression characterizes a biologically and clinically aggressive breast cancer subtype. In this study, 60 samples from Iraqi women with breast cancer were collected and investigated for HER2 protein in the tissue by immunohistochemistry. Also, 20 samples from healthy Iraqi women were used as a control. The results showed that 18 (30 %) patients expressed the HER2 protein. A molecular study for single nucleotide polymorphism (SNP) was conducted on samples metastasizing to lymph nodes. DNA was extracted and polymerase chain reaction (PCR) was performed to amplify exon 17 and intron 17 of HER2 gene. Sequencing of PCR product was achieved and two SNPs of HER2 gene, one in exon 17 (Ile655Val) and another close to it in intron 17 (rs903506) were studied. In exon 17, SNP Ile655Val was found in 41% of patients, while in intron 17, the non-coding SNP rs903506 was found in 27% of patients. However, no polymorphism was found in the control group. The results may suggest that HER2 gene can be used as a molecular marker for breast cancer.

A new primer set for amplification of ITS-rDNA in Ditylenchus destructor

A technique was developed for the identification of Ditylenchus destructor nematode belonging to the Ditylenchus genus, based on the use of different primers for polymerase chain reaction (PCR). Two universal ribosomal primers were amplified to the internal transcribed spacer region ITS-rDNA. The sequencing of PCR products confirmed the polymorphism between species. The primers were sensitive to generate a particular band of the correct size (300bp) from the DNA template of a single, separate D. destructor stage of development. Screening populations of D. destructor from Iran and the Russian Federation have tested the reliability of the primers, and the expected size of the band was produced for all test populations. Ditylenchus destructor closely related species have also been tested and no specific band was amplified. Such results showed that the primers currently developed are useful for quantifying the D. destructo r density in potato tuber.

Natural phytoplasma infections on fruit, vegetable and weed plants at the same agroecosystem and their molecular properties

The phytoplasma associated diseases are an emerging threat to fruit and vegetable crops leading severe yield losses worldwide. Pear (Pyrus communis L.) trees, with symptoms of severe reddening, dwarfing and shoot proliferation were observed in pear orchards of Malatya province of Turkey. Tomato (Solanum lycopersicum L.) plants grown nearby the symptomatic pear orchard displaying leaf rolling, severe flower sterility and purple leaves were observed at the same agroecosystem. To verify the presence and diversity of phytoplasmas, symptomatic pears and tomatoes were sampled and weeds nearby the symptomatic plants were collected. Total plant DNA was purified from midrib of collected leaves using a commercial kit. The DNA samples were analyzed by nested polymerase chain reaction (PCR) using universal primer pairs to amplify 16S rDNA fragments. The phytoplasmas detected in collected samples were differed according to the host. Here we detected and characterized ‘Candidatus Phytoplasma pyri’ belonging to apple proliferation group (subgroup 16SrX-C) from a pear tree, ‘Candidatus Phytoplasma trifolii’ belonging to clover proliferation group (subgroup 16SrVI-A) from a weed (Amaranthus retroflexus) and ‘Candidatus Phytoplasma solani’ belonging to the stolbur phytoplasma group (subgroup 16SrXII-A) from a tomato plant. Direct sequencing of PCR products verified the phytoplasmal nature of the infections. The occurrence of ‘Ca. P. trifolii’ on A. retroflexus is the first report for the world. The irregular presence of the phytoplasmas in fruit and vegetable crops and weeds indicates continuous spread of the phytoplasmas threatening the new crops and new horizons.

Molecular characterization of metallo-\u03b2-lactamase- producing carbapenem-resistant Enterobacter cloacae complex isolated in Heilongjiang Province of China

BackgroundEnterobacter cloacae complex (ECC) is one of the most common extended-spectrum β-lactamase and carbapenemase-producing pathogen that threatens millions of the elderly and vulnerable sick persons. The objective of this study was to perform the molecular characteristics of the carbapenem-resistant E. cloacae complex (CREC) emerged in Heilongjiang Province of China.MethodsSix CREC strains were isolated from the patients with infectious diseases. The identities of ECC isolates were confirmed by sequencing the polymerase chain reaction (PCR) products of 16S rRNA gene. The characterization of the CREC isolates were analyzed by sequencing PCR products of the carbapenemase, ampC and fluoroquinolone resistance genes and performing multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome sequencing.ResultsAll 6 isolates harbored multiple resistance genes. Of them, 5 carried metallo-β-lactamases and one was blaKPC-2-positive. The levofloxacin and ciprofloxacin-resistant strains had substitutions of gyrA83, gyrA87, and parC80 in the quinolone-resistance determining regions. The MLST analyses revealed that 6 isolates belonged to five sequence types (ST520, ST528, ST1119, ST1120, and ST93) while the PFGE patterns of the isolates fallen into four clusters. The strain ST1120 was found to carry two separated plasmids that encode blaNDM-1 and blaIMP-4.ConclusionsOur study, for the first time, identified a CREC strain that co-produces blaNDM-1 and blaIMP-4 in the Northeast China. Our finding emphasizes an urgent need for more intensive surveillance and precaution measures to prevent the CERC spread.

Single Nucleotide Polymorphism on Exon 2 Leptin Gene of Pasundan Cattle

Single Nucleotide Polymorphism on exon 2 of leptin gene which changes encoding from Arginine to Cysteine could change the function of Leptin as an obese gene which regulated feed intake and energy expenditure. The aims to analyze point mutation based on SNP exon 2 of Leptin gene of Pasundan cattle. This research used 49 DNA of Pasundan cattle from BPPT SP Cijeungjing. Multiplication of DNA was done by Polymerase Chain Reaction (PCR). Analyze of SNP on exon 2 Leptin gene based on nucleotide sequence from direct sequencing of PCR product which was alligned with leptin gene sequence from gene bank using Bioedit and MEGA 5.2 program. The results showed nucleotide sequence of exon 2 Leptin gene in Pasundan cattle is different from Leptin gene sequence of Bos indicus and Bos taurus. It was found one synonymous SNP that did not change amino acids Serine encoding on S17S, and two non-synonymous SNP which altered amino acids encoding, i.e R25C and R25H. In Pasundan cattle, the frequency of C allele (42.86%) was higher than A allele (31.64%) and T allele (25.50%). Six genotypes were identified i.e. CC (24.49%), CT (32.65%), CA (4.08%), TT (8.16%) and TA (2.05 %), AA (28.57%).

Incidence and association of TCF7, TCF7L2 single nucleotide polymorphisms in type 1 diabetes mellitus patients of South Tamil Nadu, India

Background: The TCF family genes TCF7 (T cell specific transcription factor-7) and TCF7L2 (transcription factor 7 like 2) are increasingly recognized to play a pivotal role in the incidence, pathophysiology of type 1 diabetes mellitus (T1DM). However, the prevalence and the influence of these allelic variants in the Indian/south Indian T1DM population is completely obscure.Methods: Genomic DNA was isolated from the peripheral blood samples of healthy controls, T1DM patients, and PCR (polymerase chain reaction), restriction fragment length polymorphism (RFLP), allele specific PCR (ASP), PCR product sequencing strategies were utilized to determine the prevalence of the TCF7 (exon 3, flanking intron 2, 3 regions) and TCF7L2 (intron 4) polymorphisms. Clinical investigations included assessment of the blood glucose/ estimated average glucose levels (EAG) and C-peptide levels.Results: The results indicate that 34.9% and 3.17% of the T1DM patients harbored the TCF7L2 rs7903146 and the TCF7 rs386692598 polymorphisms, respectively. Assessment of biochemical parameters indicated that the rs7903146 positive T1DM patients exhibited significantly lower EAG levels (p&lt;0.05), suggesting that these patients may exhibit phenotypic heterogeneity, a milder disease course. The study further demonstrates that PCR based strategies enable reliable molecular diagnosis of T1DM in small scale diagnostic units.Conclusions: T1DM patients from south Tamil Nadu present TCF7, TCF7L2 genetic variations and screening for these polymorphisms will empower physicians to provide appropriate therapy and genetic counselling.

Sequencing and Phylogenetic Analysis of the Salmonella Enterotoxin (stn) Gene of Salmonella spp. Isolated from Egyptian Broiler Breeder Chickens Farms

Salmonella is a member of Enterobacteriaceae family that found to be pathogenic to domestic and wild animals and humans. Salmonellae were isolated from three distinct governorates, Giza, Monofia and Qaluobia from broiler breeder chicken farms. Molecular characterization of the Salmonella isolates using polymerase chain reaction (PCR) assay as well as Sequencing and phylogenetic analysis of PCR products were conducted to distinguish the collected Salmonellae species. The nucleotide sequence of 617 bp PCR products representing the amplified fragment of stn gene of seven isolates of Salmonella enteritidis has been sequenced. Furthermore, the nucleotide sequence was submitted to the gene bank. The obtained sequences were blasted with the highly similar sequences and the multiple sequence alignments were conducted. Neighbor-joining tree was constructed for the Egyptian Salmonella isolates against 30 Salmonella spp. from the Gene bank database representing maximum similarity with stn gene when subjected to multiple sequence alignment, and the phylogenetic tree was constructed based on the comparative analysis of related sequences at the nucleotide level.

Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections.

Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.