Restriction Fragment Length Polymorphism Analysis Research Articles

Molecular Identification, Pathogenesis, and Life Cycle of Sarcocystis cruzi from Cattle (Bos taurus) in New Valley Governorate, Egypt.

Sarcocystis cruzi was identified by molecular methods from an intermediate host, cattle (Bos taurus), in El-Kharga, New Valley Governorate, Egypt, and its life cycle and pathogenicity were studied in the final host, dogs (Canis familiaris). 600 slaughtered cattle aged 6-8 years (480/120 males/females) were included. In addition, three laboratory-bred, coccidian-free puppies aged 2-3 months were fed infected bovine muscles to locate the definitive host and analyze sporogony. 18S rRNA-specific gene primers were used for DNA amplification from esophageal muscles. These polymerase chain reaction (PCR) amplicons were subjected to restriction fragment length polymorphism (RFLP) and molecular sequence analysis. Infection was detected in 78.8% (473/600; 95% CI, 75.56-82.11%). Histopathological examination of esophageal muscles showed oval- to spherical-shaped cysts, 96.7 μm wide by 326.9 μm long; cysts in cardiac muscles were ovoid and smaller. Infected puppies began shedding sporocysts in feces 7 days post-inoculation and showed distorted organ architecture, severe cellular damage, and inflammatory lesions in liver, kidney, esophagus, and stomach. Three oocysts with different shapes and sizes were identified. Partial 18S rRNA gene sequences of isolated New Valley sarcocysts were identical to S. cruzi isolated from different areas, verifying their genetic relatedness. Our analysis suggests that S. cruzi is the most prevalent in slaughtered cattle in New Valley Governorate, Egypt.

Sub-microscopic Plasmodium falciparum parasitaemia, dihydropteroate synthase (dhps) resistance mutations to sulfadoxine–pyrimethamine, transmission intensity and risk of malaria infection in pregnancy in Mount Cameroon Region

BackgroundPlasmodium falciparum resistance to intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) continues to spread throughout sub-Saharan Africa. This study assessed the occurrence of microscopic and sub-microscopic P. falciparum parasitaemia, dihydropteroate synthase mutations associated with resistance to SP and maternal anaemia in the Mount Cameroon area.MethodsConsenting pregnant women living in semi-rural and semi-urban/urbanized settings were enrolled in this cross-sectional study. Socio-demographic, antenatal and clinical data were documented. Microscopic and sub-microscopic parasitaemia were diagnosed using peripheral blood microscopy and nested polymerase chain reaction (PCR) respectively. The dhps mutations were genotyped by restriction fragment length polymorphism analysis. The presence of A437G, K540E, and A581G was considered a marker for high-level resistance. Haemoglobin levels and anaemia status were determined.ResultsAmong the women, the prevalence of microscopic and sub-microscopic P. falciparum infection were 7.7% (67/874) and 18.6% (93/500) respectively. Predictors of microscopic infection were younger age (< 21 years) (AOR = 2.89; 95% CI 1.29–6.46) and semi-rural settings (AOR = 2.27; 95% CI 1.31–3.96). Determinants of sub-microscopic infection were the rainy season (AOR, 3.01; 95% CI 1.77–5.13), primigravidity (AOR = 0.45; 95% CI 0.21–0.94) and regular ITN usage (AOR = 0.49; 95% CI 0.27–0.90). Of the145 P. falciparum isolates genotyped, 66.9% (97) carried mutations associated with resistance to SP; 33.8% (49), 0%, 52.4% (76) and 19.3% (28) for A437G, K540E, A581G and A437G + A581G respectively. The A581G mutation was associated with ≥ 3 SP doses evident only among sub-microscopic parasitaemia (P = 0.027) and multigravidae (P = 0.009). Women with microscopic infection were more likely from semi-rural settings (AOR = 7.09; 95% CI 2.59–19.42), to report history of fever (AOR = 2.6; 95% CI 1.07–6.31), to harbour parasites with double resistant mutations (AOR = 6.65; 95% CI 1.85–23.96) and were less likely to have received 2 SP doses (AOR = 0.29; 95% CI 1.07–6.31). Microscopic infection decreased Hb levels more than sub-microscopic infection.ConclusionThe occurrence of sub-microscopic P. falciparum parasites resistant to SP and intense malaria transmission poses persistent risk of malaria infection during pregnancy in the area. ITN usage and monitoring spread of resistance are critical.

ABCB1 gene variants as risk factors and modulators of age of onset of demyelinating disease in Mexican patients.

The C1236T, G2677T/A, and C3435T variants of the ABCB1 gene alter the functioning of P-glycoprotein and the transport of endogenous and exogenous substances across the blood-brain barrier, and act as risk factors for some neurodegenerative diseases. This study aimed to determine the association between demyelinating disease and the C1236T, G2677T/A, and C3435T variants of ABCB1 and its haplotypes and combinations of genotypes. Polymerase chain reaction with restriction fragment length polymorphism analysis (PCR-RFLP) and Sanger sequencing were used to genotype 199 patients with demyelinating disease and 200 controls, all Mexicans of mixed race; frequencies of alleles, genotypes, haplotypes, and genotype combinations were compared between patients and controls. We conducted a logistic regression analysis and calculated chi-square values and 95% confidence intervals (CI); odds ratios (OR) were calculated to evaluate the association with demyelinating disease. The TTT and CGC haplotypes were most frequent in both patients and controls. The G2677 allele was associated with demyelinating disease (OR: 1.79; 95% CI, 1.12-2.86; P = .015), as were the genotypes GG2677 (OR: 2.72; 95% CI, 1.11-6.68; P = .025) and CC3435 (OR: 1.82; 95% CI, 1.15-2.90; P = .010), the combination GG2677/CC3435 (OR: 2.02; 95% CI, 1.17-3.48; P = .010), and the CAT haplotype (OR: 0.21; 95% CI, 0.05-0.66; P = .001). TTTTTT carriers presented the earliest age of onset (23.0 ± 7.7 years, vs 31.6 ± 10.7; P = .0001). The GG2677/CC3435 genotype combination is associated with demyelinating disease in this sample, particularly among men, who may present toxic accumulation of P-glycoprotein substrates. In our study, the G2677 allele of ABCB1 may differentially modulate age of onset of demyelinating disease in men and women.

Effect of FASN, SCD, and GH Genes on Carcass Fatness and Fatty Acid Composition of Intramuscular Lipids in F1 Holstein × Beef Breeds

To improve beef quality, a selection of specific breeds for crossbreeding, genotyping, and selection of specific candidate genes in breeding animals can be some of the solutions. The objective of this study was to determine the effects of FASN, SCD, and GH genes on carcass fatness and fatty acid (FA) composition of intramuscular lipids of crossbred Holstein × beef breeds (Simmental, Belgian Blue, Limousin, and Piemontese). The allelic and genotypic distribution of polymorphisms in the FASN, SCD, and GH genes was studied in 80 crossbreed animals. Genomic DNA was isolated from musculus longissimus dorsi, whose chemical composition was determined by near infrared transmittance spectrophotometry, while the fatty acid composition was determined by gas chromatography. DNA polymorphism was analyzed by restriction fragment length polymorphism analysis. The FASN (g. 17924A&gt;G) polymorphism was significantly associated with C19:1 n-9 and C24:1 n-9, whereas GH (g.2141C&gt;G) was significantly associated with C16:0 and C20:1 n-9. The SCD (g.8586C&gt;T) polymorphism was significantly associated with C16:0, C18:0, C20:0, C14:1 n-5, C16:1, C18:1, C18:2 n-6, C18:3 n-3, C20:2 n-6, and C20:4 n-6, and analyzed the sum and ratios of fatty acids. Sex had significant effect on carcass fatness and fatty acid composition. This study provided useful results for the above candidate genes and their association with some FA, supporting their influence as genes associated with fats and fatty acid composition in beef meat.

The association of paraoxonase I gene polymorphisms Q192R (rs662) and L55M (rs854560) and its activity with metabolic syndrome components in fars ethnic group.

Metabolic syndrome (MetS) may cause premature development of some diseases. PON1 genes may be involved in the pathogenesis of MetS. The aim of study was to evaluate the association between Q192R and L55M gene polymorphisms and its enzyme activity with the MetS components in subjects with and without MetS. Polymerase Chain Reaction and Restriction Fragment Length Polymorphism analysis were performed to determine polymorphisms of the paraoxonase1 gene in subjects with and without metabolic syndrome. Biochemical parameters were measured by spectrophotometer. The MM, LM, and LL genotype frequencies of the PON1 L55M polymorphism were 10.5, 43.4, and 46.1%, and 22.4, 46.6, and 31% and; the QQ, QR, and RR genotype frequencies of the PON1 Q192R polymorphism were 55.4, 38.6 and 6%; and 56.5, 34.8 and 8.7% in subjects with and without MetS, respectively. The L and M allele frequencies were 68 and 53%; and 32 and 47% for PON1 L55M in subjects with and without MetS, respectively. The Q and R allele frequencies for PON1 Q192R were 74 and 26% in both groups. There were significant differences in HDL-cholesterol level and PON1 activity in the genotypes QQ, QR, and RR of the PON1 Q192R polymorphism in subjects with MetS. The PON1 Q192R genotypes had only effected on PON1 activity and HDL-cholesterol level in subjects with MetS. Different genotypes of the PON1 Q192R seem to be important candidates to make the subjects susceptible to MetS in the Fars ethnic group.

The SOCS-1 -1478CA/del functional polymorphism (rs33989964) is associated with gastric cancer but is unrelated to overall survival.

In vitro studies have shown that the functional - 1478CA > del polymorphism (rs33989964) of the suppressor of cytokine signaling (SOCS)-1 gene is associated with an altered trascriptional activity. Here, we sought to examine the potential association of this polymorphism with the risk of gastric cancer (GC) and to analyze its prognostic impact on overall survival (OS). The study cohort consisted of 74 Turkish patients with GC and 52 healthy controls. Genotyping of the SOCS-1 -1478CA > del polymorphism was carried out using restriction fragment length polymorphism analysis. After allowance of age and sex, multivariable logistic regression analysis revealed that the carriage of the del allele of the SOCS-1 -1478CA > del polymorphism was independently associated with an increased risk of GC (odds ratio = 6.78, 95% confidence interval = 3.24-10.99, P < 0.001). Kaplan-Meier analysis revealed no significant differences in OS for patients harboring at least one del allele of rs33989964 compared with CA/CA homozygotes (log-rank test, P = 0.17). While the SOCS-1 -1478CA > del polymorphism is significantly associated with the risk of GC in the Turkish population, it does not affect OS.

Profile of Gut Bacteria in Hypertensive Patients Based on Terminal Restriction Fragment Length Polymorphism Analysis

Hypertension is a severe public health problem due to its high prevalence worldwide. About 7.5 million deaths or 12.8% of all annual deaths worldwide occur due to high blood pressure. The hypertensive disease is also associated with the intestinal bacteria. To our knowledge, the diversity of the gut bacteria in hypertensive patients has not been reported yet in Indonesia. Thus, the aims of this study were to analyze profile of gut bacteria in hypertensive patients compared to non-hypertensive based on metagenomic analysis, Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results of the affiliation analysis of the entire Terminal Restriction Fragments (TRF) contained 6 groups of bacteria from 26 TRFs in hypertensive and non-hypertensive respondents. Cutting the 16S rRNA gene with the BslI restriction enzyme successfully detected intestinal bacterial groups, namely Clostridium subcluster XIVa, Prevotella, Clostridium cluster IV, Clostridium cluster XI, Bacteroides and others. In hypertensive patients, a higher relative abundance of bacterial groups showed in Clostridium cluster XI, Clostridium cluster IV and Clostridium subcluster XIVa. The abundance of Bacteroides and Prevotella groups in hypertensive patients were lower than non-hypertensive. The abundance of enterotype I and enterotype II was lower in hypertensive patients than non-hypertensive. Contrarily to that enterotype III cluster. It is worth noting that the F/B ratio was higher in hypertensive patients than that in non-hypertensive. Our data suggest that the gut bacteria profile of hypertensive patients differs to that non-hypertensive.

First report of 16SrII-V phytoplasma associated with cowpea (Vigna unguiculata) phyllody in China

Cowpea plants exhibiting virescence, phyllody and little leaf symptoms were observed in the fields of Zhanjiang City, Guangdong province of China in October 2020. Total DNA was extracted from symptomatic cowpea leaves. Using phytoplasma universal primer pairs (R16mF2/R16mR1, P1/P7) and a secA primer pair (secAfor1/secArev3), expected PCR fragments of approximately 1400, 1800 and 800 bp were amplified from all symptomatic cowpea leaves. MUSCLE analysis revealed that the representative 16S rRNA gene sequence of cowpea phyllody phytoplasma (CPP) shared 98.67% nucleotide identity with the reference strain WBDL of ‘Ca. Phytoplasma aurantifolia’ (GenBank accession number U15442); and 100% nucleotide identity with 16SrII-V phytoplasmas associated with ‘Praxelis clematidea’ phyllody (GenBank accession number KY568717); ‘Desmodium triflorum’ little leaf (GenBank accession number MT452308); and ‘Ixeris chinensis’, ‘Emilia sonchifolia’, and ‘Desmodium ovalifolium’ witches’ broom (GenBank accession number MT416114, MT420682 and MK956144, respectively). Phylogenetic analyses based on the 16S rRNA and secA gene sequences revealed that the CPP strain clustered with the 16SrII-V subgroup and 16SrII group, respectively. Virtual restriction fragment length polymorphism analysis of the 16S rRNA sequence revealed that CPP was associated with the 16SrII-V subgroup. To our knowledge, this is the first report of the16SrII-V subgroup of phytoplasmas associated with cowpea phyllody disease.

The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice

Endophytic persistence of inoculated plant growth promoting bacteria (PGPB) involves interaction with the host plant and the host’s indigenous endophytic bacterial communities. This study investigated the persistence of Methylobacterium oryzae CBMB20 into the rice endosphere together with the impact of inoculation on the diversity and community structure of the root and shoot bacterial endophytes in Oryza sativa L. spp. indica cv. IR29. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of the root and shoot showed that M. oryzae CBMB20 was able to integrate and persist in the rice endosphere without causing drastic shifts in bacterial endophytic diversity and community composition. The bacterial communities in the root and shoot are very similar to the seeds of IR29, suggesting that most of them are seed-borne. The root endosphere bacterial communities of inoculated and uninoculated IR29 plants are more diverse compared to the shoots in terms of richness and diversity indices. The dominant bacterial T-RFs of the root endosphere of IR29 belong to Microbacterium, Delftia, Pseudomonas, Xanthomonas and Stenotrophomonas, Herbaspirillum, Enterobacter, and Sphingomonas, as observed in the three restriction enzyme T-RFLP profiles. Bacterial clades identified as Curtobacterium, Enterobacter, Stenotrophomonas, and Xanthomonas were distinctly observed in both the root and shoot communities, and these bacterial groups are also the dominant endophytes of the shoot endosphere. This study showed that Methylobacterium oryzae CBMB20 could persist and incorporate into the endophytic bacterial community of the endosphere without causing long-term antagonistic interactions with its host plant and with the native microbiota.

Detection and molecular identification of a 16SrI group phytoplasma associated with sisal purple leafroll disease

Sisal purple leafroll disease (SPLD) is a destructive disease affecting sisal in China, however, its etiology remains unknown. This disease is characterized by purple margins and rolling and yellowing of the leaves. Recently, phytoplasma was found to be present in SPLD-affected sisal according to PCR assay. To further verify the presence of phytoplasma, determine its association with SPLD and identify the possible pathogenic phytoplasma infecting sisal, 80 symptomatic and 65 asymptomatic sisal samples were collected from the main sisal farms in three Chinese provinces, including Hainan, Guangdong and Guangxi provinces, from 2018 to 2021. PCR assays and transmission electron microscopy further revealed the presence of phytoplasma. Nested PCR using the universal primer R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasma in sisal. A comparison of the detection rate of phytoplasma in symptomatic (83%) versus asymptomatic (17%) sisal populations showed some association between phytoplasma and SPLD. Sequencing analyses, phylogenetic analyses, and virtual restriction fragment length polymorphism analyses confirmed the presence of phytoplasma in sisal belonging to 16SrI-B. This is the first report of 16SrI-B phytoplasma infecting sisal in China.

First Molecular Identification of Fasciola gigantica in Slaughtered Cattle in Cape Verde: Prevalence, Gross Pathological Lesions, Genetic Identification and Coprological Analysis.

A study on fasciolosis prevalence, gross pathological lesions, fluke genetic identification and coprological analysis was carried out in slaughtered cattle from one abattoir in Cape Verde. Of the 131 cattle inspected over two months, 12 (9.0%) presented fasciolosis-compatible lesions (FCL) that resulted in liver condemnation. The genetic characterization of the flukes collected, through restriction fragment length polymorphism analysis of PCR-amplified fragments (PCR-RFLP), confirmed the presence of Fasciola gigantica; therefore, being the first identification of this species in cattle from Cape Verde. Animals that released Fasciola spp. eggs and, thus, responsible for environment contamination (positive shedders), were identified through coprological analysis (natural sedimentation technique). Of the 12 animals with FCL, samples from 11 were submitted to coprological analysis and 7 (63.6%) were found to be positive shedders. Furthermore, of the 82 animals with non-FCL, randomly selected for coprological analysis, 4 (4.9%) were also found to be positive shedders for Fasciola spp. The results of this study, regarding species identification and coprological analysis, are epidemiologically important to update the information regarding fasciolosis in Cape Verde. The new data could help implement effective strategies for disease control and mitigation, consequently reducing economic loss and the level of animal and human infection from the One Health perspective.

Association of C677T Polymorphism of Methylenetetrahydrofolate Reductase with Metabolic Syndrome among Eastern Indian Women: A Case-control Study

Introduction: Polymorphisms of Methylene Tetrahydrofolate Reductase (MTHFR) gene have been associated with hyperhomocysteinaemia, which in turn may lead to hypertension, insulin resistance and abnormality of lipid metabolism. All these abnormalities are also components of Metabolic Syndrome (MetS). Thus, there is possibility of an association between MetS and MTHFR polymorphism. Literature search revealed paucity of data on this association, particularly from India. Aim: To evaluate the association of C677T polymorphism of MTHFR gene with MetS, among women of eastern Indian population. Materials and Methods: A hospital-based case-control study was conducted in the Department of Biochemistry, Calcutta National Medical College, Kolkata, West Bengal, India, between December 2016 to June 2018. Anthropometric and biochemical profiling of all 417 study subjects were done. Genetic workup was done by Polymerase Chain Reaction (PCR), using suitable primer, followed by restriction fragment length polymorphism analysis using Hinf 1 enzyme to identify MTHFR C677T polymorphism. Subjects were divided into two groups according to presence or absence of MetS as per International Diabetic Federation worldwide definition and compared with Mann-Whitney U Test. Logistic regression analysis was also performed. Results: Out of enrolled 417 female subjects, 243 were categorised as MetS group. Mean age (in years), of MetS group (48.70±9.03) was found to be significantly higher (p&lt;0.001) than that of control group (42.83±10.90). A total of 45 (25%) females with MetS exhibited the presence of heterozygous CT genotype; that was significantly higher (p=0.026) than the non MetS group 21 (14.9%). The mutant T allele frequency was also significantly higher among the subjects suffering from MetS; 45 (12.5%)compared to 21 (7.4%) in the control group (p=0.036). Multiple logistic regression analysis revealed the odds of developing MetS among subjects with heterozygous CT genotype was 7.721 times (CI: 2.38-25.05) compared to those with wild genotype. Conclusion: C677T polymorphism of MTHFR gene was associated with the occurrence of MetS among woman.

Modification of a previously patented method to unequivocally score A2-like and A1-like bovine β-casein variants

A growing interest in the production and commercialization of A2 cow's milk has been observed in many countries in the last few years due to the beneficial properties for human health attributed to A2 β-casein variant. Methods of varying complexity and different equipment requirements have been proposed for the determination of the β-casein genotype of individual cows. We proposed herein a modification of a previously patented method based on an amplification-created restriction site PCR followed by restriction fragment length polymorphism analysis. This method allows to identify and differentiate A2-like from A1-like β-casein variants, after differential endonuclease cleavage flanking the nucleotide that determines the amino acid at position 67 of β-casein. The advantages of this method are that it:• enables to unequivocally score A2-like as well as A1-like β-casein variants,• can be performed at low cost in simply equipped molecular biology laboratories, and• can be scaled up to analyze hundreds of samples per day.For these reasons, and based on the results obtained from the analysis carried out in this work, it showed to be a reliable method for the screening of herds to selective breeding of homozygous cows and bulls for A2 or A2-like alleles.

Comparison of different DNA preparatory methods for development of a universal direct PCR-RFLP workflow for identification of meat origin in food products

The quality and quantity of the extracted DNA are two key aspects for a successful PCR (Polymerase Chain Reaction) amplification. Moreover, reduction in time and cost required for DNA extraction are also two considerable factors, in cases when large number of samples are to be analyzed within a limited time-frame and budget. Accordingly, the aim of this study was to compare and optimize performance of five different DNA extraction methods by boiling meat tissues from cattle, buffalo, sheep, goat, chicken, camel, horse and dog in PBS (Phosphate Buffer Saline), distilled water, alkaline lysis buffers 1, 2 or 3. The results indicated that the boiling of meat and its products in alkaline lysis buffers was a good method to extract crude DNA. The optimized crude DNA extraction protocol was coupled with PCR-RFLP (Restriction Fragment Length Polymorphism) analysis for meat species differentiation. This developed workflow was tested on fifty-three commercial beef and mutton samples, out of which three samples were found to be adulterated. In conclusion, the rapid crude DNA extraction protocol has led to the development of a direct PCR-RFLP workflow that is simple, time-saving and cost-effective for PCR-based identification of different meat species.

Association of rs4646903 and rs1048943 CYP1A1 estrogen-metabolizing gene polymorphisms with estrogen receptor-positive breast cancer in Kenyan women

Breast cancer is the most prevalent neoplasm and the second leading cause of death among females in Kenya. Estrogen and its metabolites are known risk factors for breast cancer. Polymorphisms in these genes and breast cancer susceptibility are unique among different populations. This study aimed to determine the probable associations between estrogen-metabolizing gene variations and other risk factors for breast cancer risk in Kenyan women. Buffy coat samples were obtained from patients diagnosed with estrogen receptor-positive breast cancer, benign breast disease, and healthy volunteers. Genotyping of target polymorphisms was conducted using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The rs4646903 variant genotype CC was associated with breast cancer in the case-control model (P=0.001); the heterozygous genotype TC (P=0.01) and the luminal B molecular subtype (P=0.02) showed increased odds of late-stage breast cancer. The rs1048943 variant genotype GG was associated with breast cancer in the case-benign model (P=0.04), whereas CG was associated with breast cancer in the case-control model (P=0.02). These findings imply that the rs4646903 and rs1048943 variant genotypes are involved in breast cancer risk in Kenyan women. Hence, they may be explored further as potential markers for the disease.

Current Status of Phytoplasma Diseases in the Vineyards of Eastern and Southeastern Anatolia Regions

Doğu ve Güneydoğu Anadolu Bölgesinde yer alan Adıyaman, Batman, Elazığ, Malatya, Mardin, Şanlıurfa ve Diyarbakır illerindeki bağ alanlarında fitoplazma hastalık etmenlerinin durumlarını belirlemek için 2013-2021 yılları arasında sörveyler gerçekleştirilmiştir. Güdümlü örnekleme yöntemine göre yapılan bu sörveyler sonucu 1110 bitki örneği toplanmıştır. Fitoplazma varlığını belirlemek amacıyla toplanan örneklerin Gerçek Zamanlı Polimeraz Zincir Reaksiyonu (qPCR) analizi ile hızlı teşhisleri gerçekleştirilmiştir. qPCR analizi sonucunda biri Elazığ diğeri ise Mardin ilinde olmak üzere toplam 2 örnekte fitoplazma etmeni tespit edilmiştir. Tespit edilen pozitif örneklerin teşhisleri için R16mF2/R16mR1 ve R16F2n/R16R2 üniversal primerlerinin kullanıldığı Yuvalanmış Gerçek Zamanlı Polimeraz Zincir Reaksiyonu (Nested qPCR) analizleri gerçekleştirilmiş ve 1.25 kb boyutunda bantlar elde edilmiştir. Fitoplazma örneklerinin 16S rDNA bölgesinden elde edilen 1.25 kb’lik amplifikasyon ürünlerine BLAST ve sanal RFLP analizleri uygulanmıştır. Bu analizler ile doğrulanan 2 pozitif örnekte ‘Candidatus Phytoplasma solani’ (16Sr group XII, subgroup A)’nin varlığı belirlenmiştir (OM212474 ve OM909048). Fitoplazma izolatlarına ait 16S rDNA dizilerinin BLAST karşılaştırmasında dünyadaki diğer fitoplazma izolatları ile %99-100 arasında benzerlik gösterdiği tespit edilmiştir.

Breeding characteristics of Romanov sheep depending on polymorphism of growth hormone gene

The search for new markers of high productivity of sheep and the introduction of evaluation and selection methods for these indicators is an urgent direction in breeding of Russian sheep. The material for the research was the results of assessment of exterior, live weight of Romanov sheep. Genetic studies were carried out using tissue samples (ear plucking). Polymorphism of growth hormone gene in Romanov sheep was evaluated by restriction fragment length polymorphism analysis and polymerase chain reaction. Evaluation of polymorphism of growth hormone gene in the population of Romanov sheep showed the presence of three allelic variants - AA, AB and BB, with a frequency of 0.24, 0.63 and 0.14, respectively. The statistically significant difference between the indicators of live weight in animals with genotypes GHAB and GHAA, GHAB and GHBB was: at birth 210 g and 140 g (P 0.95), respectively; at weaning 350 g and 1260 g (P 0.95), respectively. The phenotypic variability of live weight was the highest at birth and at weaning and amounted to 31.732.5 % for all genotypes. Animals with genotypes GHAA and GHAB outperformed their peers with genotype GHBB in dimensions of rump width in tuber coxae, rump width in tuber trochanterica, chest width, chest circumference, and therefore, had a wide body. When studying the phenotypic and genetic correlations of measurements of exterior and live weight at the age of 12 months, a reliable positive relationship was established with height at withers height (rP = +0.13*; rG = +0.02), rump height (rP = +0.14**; rG = +0.03), rump length (rP = +0.14**; rG = +0.02). The new knowledge gained about polymorphism of GH gene and its relationship with productive traits of Romanov sheep will allow for in-depth evaluation, targeted selection and selection of individuals with desirable genotypes.

Molecular Characterization and Gene Expression Of TLR4 Gene Associated with Mastitis in Goats

In this study characterization of Toll-like receptor 4 (TLR4) gene of goats; detection of polymorphisms in the nucleotides, and determination of the association of identified genotypes with the occurrence of subclinical mastitis was done using chi-square and odds ratio. Analyzing gene expression using two-sided Student's T-test was also done. Results of Study 1 revealed high similarity (99%) of TLR4 nucleotide sequence of all breeds of goats with that of C. hircus (NM_001285574.1) and sheep (Ovis aries) sequences and slightly lower similarity with cattle (Bos taurus, and Bos indicus) (96%), and water buffalo (Bubalus bubalis) (95%). In Study 2, restriction fragment length polymorphism (RFLP) analysis revealed three genotypes with nine restriction patterns using AluI enzyme. Genotype AA has odds ratio of 0.28 and 0.08 in all breeds, and in Anglo-Nubian, respectively, with significant association (P0.05) that inferred 0.28 and 0.08 times greater probability in all breeds, and in Anglo-Nubian, respectively, for subclinical mastitis to occur than those of other genotypes. Genotype AB showed odds ratio of 3.83, 13.00 and 2.40 in all breeds, in Anglo-Nubian, and in Upgraded, respectively, with significant association (P0.05) that indicated 3.83, 13.00 and 2.40 times more likely in all breeds, in Anglo-Nubian, and in Upgraded, respectively, to suffer subclinical mastitis than those of other genotypes. Â In Study 3, genetic expression analysis showed a significant upregulation of TLR4 gene up to maximum of 3.63-fold in goats without subclinical mastitis compared to subclinically mastitic animals with only 0.65-fold which suggest a prompt role of TLR4 gene in the protection of animal against possible infection.Â

New splice site mutations in MYO7A causing Usher syndrome type 1: a study on a Chinese consanguineous family.

This study investigated the new splice site mutations of Myosin VIIA (MYO7A) in patients with Usher syndrome type 1 (USH1) from a three-generation Chinese consanguineous family. All subjects underwent comprehensive ophthalmic examinations and an audiometric test. Demographic data, family history, and peripheral blood leukocytes were collected. We performed whole exome sequencing (WES) to analyze the genomic DNA of the family. DNA sequence and restriction fragment length polymorphism (RFLP) analyses were also done. The identified genetic variants were validated by conducting polymerase chain reaction (PCR) in 100 healthy control subjects and comparing with the NCBI VARIANT database and the 1000 Genomes Project. The functional consequences were further analyzed. WES identified two new splice site mutations (c.5648G > A(rs111033215) and c.6238-1G > C) in MYO7A in two patients with USH1, i.e., the proband and her elder brother. DNA sequence and RFLP analyses showed that other members without USH1 carried only one of the two mutations. In the analysis of healthy controls, neither mutation existed. Both mutations were predicted to be damaging and were most likely associated with USH1. In the three-generation Chinese consanguineous family with USH1, c.5648G > A(rs111033215) and c.6238-1G > C mutations in MYO7A are most likely associated with the disease. Our findings expand the mutational spectrum of MYO7A, which will enhance the understanding of the genetic abnormalities in USH1 and provide more evidence for future investigations on therapeutic strategies such as precise gene replacement or gene editing.

A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening

BackgroundGenome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. ResultsHere, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1–5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene.ConclusionsWe have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.