Specific Polymerase Chain Reaction Primers Research Articles

Molecular Characterization and Cellulolytic Capacity of Chaetomium globosum Isolates

Chaetomium species are saprophytic fungi, havethe ability to degrade cellulose with hydrolytic enzymes. Hydrolytic enzymes are a cluster of enzymes, which have the flexibility of breaking down complicated molecules into smaller molecules. The objective of this study was to clarify the genetic diversity among the isolated Chaetomium population by using PCR-based methods, along with the study of their cellulolytic activities. Chaetomium spp. were isolated from twenty-eight samples of Cuminum cyminum and Pimpinella anisum, and identified morphologically and molecularly by ITS1 and ITS4 primers. The sequencing indicated that all the isolates of Chaetomium had a 99% sequence identity with Chaetomium globosum sequences from GenBank. Molecular techniques with Internal Transcribed Spacer (ITS) region sequencing and specific genes random primers polymerase chain reaction (SGRP-PCR) showed the existence of high DNA polymorphism of Chaetomium globosum. All isolates were tested for their ability of cellulases production. Nine of the thirteen isolates of Chaetomium globosumcould produce cellulases. There are variations between C. globosum isolates and the source of the isolates and their habitat do not correlate to their ability to secrete cellulases.

TNF, IL-6, and IL-10 cytokines levels and their polymorphisms in renal function and time after transplantation.

Cytokine polymorphisms can influence their plasma levels and thus affect the immune response in renal transplantation. A total of 146 renal transplant recipients (RTR) were classified into groups according to the estimated glomerular filtration rate (R1: < 60 and R2: ≥ 60mL/min/1.73m2) and time after transplantation (T1: 1 to 24, T2: 25 to 60, T3: 61 to 120, and T4: > 120months after transplantation). The polymorphisms were genotyped by single specific primer-polymerase chain reaction. IL-10 was measured by ELISA and IL-6, and TNF levels were determined using Miliplex®. A higher frequency of the - 308G allele and the - 308G/G genotype, low-producer, was observed in the R1 group compared with R2. In addition, a higher frequency of the - 308A carriers, high-producer, was found in the R2 group. However, no significant difference was observed in cytokine levels when both groups were compared. Higher levels of IL-6 were observed in T1 compared with T2 and T4 groups. Lower IL-6 levels were found in T2 compared with T3 group. Lower levels of IL-10 were also found in T1 group in relation to T2, while higher levels of this cytokine were observed in T2 group compared with T3. The results suggest that the - 308G > A polymorphism in the TNF gene is associated with filtration function after renal transplantation, and IL-6 and IL-10 levels change according to the time after transplantation. Thus, the joint evaluation of - 308G > A polymorphism in TNF gene and IL-6 and IL-10 levels would provide a broader and effective view on the clinical monitoring of RTR.

Specific PCR primers for the identification of Salmonella enterica serovar enteritidis in chicken-related samples

In this study, a designed pair of PCR primers, SefB127L-SefB661R, based on the sefb gene (accession number L11009) sequences was used in polymerase chain reaction (PCR) for rapid evaluation of Salmonella enterica serovar Enteritidis in chicken-related samples. The specificity of this method was checked with 85 Salmonella strains and 17 non-Salmonella strains. The results showed that only 24 isolates of S. enterica serovar Enteritidis exhibited 535 bp PCR product. The detection limit of this PCR method were evaluated using 40 spiked samples under enrichment protocols. The data revealed that microbial extract from as few as 101 target cells per gram of the sample culture was required for this assay. Before PCR amplification, pre-culture and the cell lysates, rather than the DNA extracts, were used directly for all tested samples. To verify the usefulness of this PCR process for S. enterica serovar Enteritidis examination, 285 endogenous samples including chicken meats, eggs and swabs of chicken-related samples and coop's facilities were tested and compared with that obtained by conventional BAM (Bacteriological Analytical Manual) method. About 1% (three in 285) of the S. enterica serovar Enteritidis samples was contaminated, approximately the same as that obtained from BAM method.

Characterization, pathogenicity and enzymatic profile of Fusarium solani associated with potato tubers in Upper Egypt

Ten strains of Fusarium solani were isolated on Potato Dextrose Agar medium from rotten potato tubers marketed in Upper Egypt region. The identification of collected isolates was carried out by morphological criteria and species-specific primer, TEF-Fs4f and TEF-Fs4r was used to confirm the identification. Genetic diversity of the collected isolates studied by using specific genes random primer polymerase chain reaction (SGRP-PCR) techniques. The results indicated that F. solani isolates have high genetic diversity and no correlation between the geographical site and banding patterns was recorded. The pathogenicity test showed that 20% of F. solani isolates registered as highly pathogenic. No resemblance was detected between pathogenicity levels and banding patterns of SGRP obtained from F. solani isolates. All F. solani isolates exhibited amylolytic, cellulolytic, pectinolytic, lipolytic and proteolytic activities and the amount of each enzyme significantly different among F. solani isolates.

Distribution of Diego blood group alleles and genotypes in the Chinese Korean population and sequence-based identification

ObjectiveTo investigate Diego blood group alleles in the Chinese Korean population. The Diego blood group system plays an important role in transfusion medicine, but the distribution of the blood group in many Chinese ethnic populations remains unclear. MethodsSequence Specific Primer Polymerase Chain Reaction (SSP-PCR) was used for Diego genotyping and sequence-based typing PCR (PCR-SBT) was used to verify single nucleotide polymorphisms in the coding region of SLC4A1 starting from exon 19. Nine hundred and ten samples from the Chinese Korean population were investigated. ResultsThe frequency of the DI*01 and DI*02 alleles in the Chinese Korean population was 0.0516 and 0.9484, respectively. The most predominant genotype was DI*02/DI*02, with a frequency of 90.22 % (821/910). The frequency of DI*01/DI*02 was 9.23 % (84/910) and that of DI*01 /DI*01 was 0.55 % (5/910). The genotype distributions of the Diego blood group conformed to the Hardy-Weinberg equilibrium (P > 0.05). ConclusionThe data obtained from this study will be helpful for the creation of a donor database to provide antigen-negative blood to patients with allo-antibodies. Genotyping can be used as a substitute for the serological technique when antisera are unavailable and is suitable for screening a large number of donors for rare-blood-group databases.

Abstract P5-01-04: Personalized monitoring of circulating tumor DNA during neoadjuvant therapy in high-risk early stage breast cancer reflects response and risk of metastatic recurrence

Abstract Background: The detection of circulating tumor DNA (ctDNA) during neoadjuvant therapy (NAT) may serve as an early indicator of emerging resistance and disease progression. In this study, we analyzed ctDNA from high-risk early breast cancer patients who received NAT and definitive surgery in the I-SPY 2 TRIAL (NCT01042379). We hypothesized that ctDNA can serve as a biomarker of response and survival in this setting. Methods: ctDNA analysis was performed on 291 plasma samples from 84 high-risk stage II and III breast cancer patients randomized either to an investigational agent MK-2206, an AKT inhibitor, in combination with paclitaxel followed by doxorubicin and cyclophosphamide (AC) (n=52)—or standard-of-care (paclitaxel followed by AC) (n=32). HER2+ patients also received trastuzumab. Serial plasma was collected at pretreatment (T0), at 3 weeks after initiation of paclitaxel treatment (T1), between paclitaxel and AC regimens (T2), and after NAT prior to surgery (T3). A personalized ctDNA test was designed to detect a set of 16 patient-specific somatic variants, initially identified from whole exome sequencing of pretreatment tumor, then tested in plasma samples. Regions containing the somatic variants were amplified from cell-free DNA using specific polymerase chain reaction primers. Amplified products were subjected to ultra-deep sequencing (mean: 94,000x) to detect somatic variants. Association between ctDNA and clinicopathologic variables was assessed using Fisher’s exact test. Association of ctDNA with response and survival was analyzed using logistic and Cox regressions, respectively. The survival endpoint of the study was distant disease-free survival. The median follow-up was 4.8 years. Results: At pretreatment (T0), 61 of the 84 (73%) patients had detectable ctDNA. Pretreatment (T0) ctDNA positivity and levels (mean mutant molecules per mL of plasma) were significantly associated with increased tumor burden (clinical T stage T3/T4), more aggressive tumor biology (higher Mammaprint scores) and subtype (HER2+ and Triple negative). CtDNA detection during NAT decreased over time (T0- 73%; T1- 35%; T2- 14%; T3- 9%). Of the 84 patients, 23 (27%) achieved pCR and all were ctDNA-negative after NAT (T3), while all 6 patients who had detectable ctDNA at T3 did not achieve pCR. Patients who cleared ctDNA early at T1 (n=27, 48% pCR rate) had significantly increased probability of achieving a pathologic complete response (pCR) compared to those who remained ctDNA-positive (n=29, 17% pCR rate; Odds ratio=4.33, Likelihood ratio p=0.012). Patients who were ctDNA-positive at T3 (n=6) had significantly increased risk of metastatic recurrence (HR 14.7; 95% CI 1.6-131.5) compared to those who achieved pCR and were ctDNA-negative (n=17). The risk of metastatic recurrence in patients who cleared ctDNA during NAT was not significantly different from those who were negative at T0 and remained negative by T3 (hazard ratio, HR: 2.1, 95% CI: 0.22-20.2). Interestingly, patients who were ctDNA-negative (n=37) but failed to achieve pCR had similar risk of metastatic recurrence with those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). Conclusions: Early clearance of ctDNA during NAT was significantly associated with increased likelihood of achieving pCR. Residual ctDNA after NAT was a significant predictor of metastatic recurrence, while clearance of ctDNA at any point during NAT was associated with improved outcomes. Taken together, personalized monitoring of ctDNA during NAT may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival. Validation studies in a larger cohort are warranted. Citation Format: Mark Jesus M Magbanua, Lamorna Brown-Swigart, Gillian Hirst, Christina Yau, Denise Wolf, Hsin-Ta Wu, Antony Tin, Svetlana Shchegrova, Himanshu Sethi, Raheleh Salari, Alexey Aleshin, Maggie Louie, Bernhard Zimmermann, Angela DeMichele, Minetta Liu, Amy Delson, Amy Jo Chien, Smita Asare, Laura Esserman, I-SPY 2 TRIAL Consortium, Laura van't Veer. Personalized monitoring of circulating tumor DNA during neoadjuvant therapy in high-risk early stage breast cancer reflects response and risk of metastatic recurrence [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-04.

Multidrug-resistant Campylobacter jejuni, Campylobacter coli and Campylobacter lari isolated from asymptomatic school-going children in Kibera slum, Kenya

Background:The objective of this study was to determine the prevalence of thermophilicCampylobacterspp. in asymptomatic school-going children and establish the antibiotic resistant patterns of the isolates towards the drugs used to treat campylobacteriosis, including macrolides, quinolones and tetracycline.Campylobacterspp. are a leading cause of enteric illness and have only recently shown resistant to antibiotics.Methods:This study isolatedCampylobacterspp., includingCampylobacter coli,Campylobacter jejuniandCampylobacter lari, in stool samples from asymptomatic school-going children in one of the biggest urban slums in Kenya. The disc diffusion method using EUCAST breakpoints was used to identify antibiotic-resistant isolates, which were further tested for genes encoding for tetracycline resistances using primer-specific polymerase chain reaction.Results:In total, 580 stool samples were collected from 11 primary schools considering both gender and age. Subjecting 294 biochemically characterizedCampylobacterspp. isolates to genus-specific PCR, 106 (18.27% of stool samples) isolates were confirmedCampylobacterspp. Out of the 106 isolates, 28 (4.83%) wereCampylobactercoli, 44 (7.58%) wereCampylobacter jejuniwhile 11 (1.89%) wereCampylobacterlari.Campylobacter jejunihad the highest number of isolates that were multi-drug resistant, with 26 out of the 28 tested isolates being resistant to ciprofloxacin (5 mg), nalidixic acid (30 mg), tetracycline (30 mg) and erythromycin (15 mg).Conclusions:In conclusion, a one-health approach, which considers overlaps in environment, animals and human ecosystems, is recommended in addressing campylobacteriosis in humans, since animals are the main reservoirs and environmental contamination is evident.

Analysis of CTLA-4 (+49A/G) Gene Polymorphism and the Risk of Pulmonary Tuberculosis in Babylon province of Iraq

Background: Pulmonary tuberculosis (pTB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (M. tuberculosis). It typically affects the lungs, but can also affect other sites (extra- pulmonary TB). The disease is spread when sick individuals expel bacteria into the air, for example by coughing.Aim of Study: To highlight the effect of CTLA-4 (+49A/G) gene polymorphism on the risk of pulmonary Tuberculosis (pTB).Patients and Methods: This case-controlled study used single specific primer-polymerase chain reaction (SSP-PCR) to analyze the CTLA-4 (+49A/G) gene polymorphism in 60 patients with pTB who were referred to consultant clinic for respiratory diseases in Hilla – Babylon province/ Iraq during the period from December 2017 to July 2018, and 60 healthy persons’ control. Blood samples were collected from both groups according to the standard methods.Results: Data analysis revealed that the frequencies of AA, AG and GG genotypes in patients were 73.33%, 23.33%, and 3.34% respectively. In controls, this frequency was 51.67%, 35%, and 13.33% respectively. Logistic regression test detected a significant difference in the frequency of the (GG genotype) mutant homozygous of this polymorphism between patients and controls (3.34% versus 13.33%), The GG genotype of CTLA-4(+49A/G) showed a significantly decreased risk of pulmonary tuberculosis disease (OR= 0.18, 95% CI= 0.04 – 0.88, P value = 0.035).Conclusion: The GG genotype of CTLA-4 may decrease the risk of pTB.

Detection of interspecific hybrids between creeping bentgrass and colonial bentgrass using DNA marker analysis

AbstractBentgrasses (Agrostis spp.) are cool‐season turfgrasses that have utility on golf courses or other turf applications requiring low mowing heights. Interspecific hybridization of Agrostis species is a strategy that has been proposed to enhance biotic and abiotic stress tolerance in this genus. Non‐transgenic, efficient, and cost‐effective methods for interspecific hybrid detection are needed. In this study two bentgrass species creeping bentgrass (A. stolonifera L.), and colonial bentgrass (A. capillaris L.) were allowed to intercross under field conditions. Progeny plant DNA was extracted and stored using Flinders Technology Associates (FTA) PlantSaver cards followed by primer specific polymerase chain reaction (PCR) and high‐resolution melt analysis. Species identifying PCR primers were optimized for real‐time PCR through fragment resequencing of species‐specific sequence characterized amplified region (SCAR) markers. A total of 2289 potential interspecific hybrid bentgrass progeny were screened with 99.4% of the DNA extractions successful. The percent interspecific hybridization, on an individual plant basis, ranged from 1 to 85% and a total of 524 interspecific‐hybrid bentgrasses were identified representing 23% of plants tested. Interspecific hybridization is common between these bentgrass species and the proposed detection method provides a non‐transgenic, efficient, and cost‐effective method for interspecific hybrid identification.

The Relationship Between Serum Concentration of Interleukin-35 and FoxP3 Polymorphism in Patients Undergoing Coronary Artery Bypass Graft Surgery.

ObjectiveTo investigate the association between interleukin-35 (IL-35) levels and single nucleotide polymorphisms (rs3761548, rs3761547) of the FoxP3 gene in coronary artery bypass grafting (CABG) patients.MethodsWe conducted a prospective study including 140 patients, who were scheduled for elective isolated on-pump CABG with cardiopulmonary bypass (CPB) from January 2017 to September 2018 in the Jorjani heart center. Blood samples were collected before and 12 hours after the operation. Serum levels of IL-35 were measured by enzyme-linked immunosorbent assay and the pattern of genetic variations was assessed using single specific primer-polymerase chain reaction.ResultsThe serum concentrations of IL-35 after surgery were significantly higher than pre-surgery levels (18.4±8.3 vs. 9.89±3.2, respectively, P=0.002). There was no significant association between genotype frequencies of rs3761548 and rs3761547 and elevated IL-35 levels (P>0.05). There were significant associations between IL-35 levels and preoperative variables, including age (r=-0.34, P=0.047) and body mass index (r=-0.41, P=0.045), and intraoperative variables, including CPB time (r=0.4, P=0.02) and mean arterial pressure (r=-0.38, P=0.046), in carriers of the rs3761548 AA genotype.ConclusionSerum IL-35 concentrations were significantly increased in CPB patients, which may contribute to the post-CPB compensatory anti-inflammatory response syndrome. IL-35 increased levels were not influenced by FoxP3 promoter polymorphisms (rs3761548, rs3761547).

IDENTIFICATION OF FOOD PATHOGENS AND DETERMINATION OF THEIR DISTRIBUTION LEVEL IN UKRAINIAN FOOD PRODUCTS OF ANIMAL AND PLANT ORIGIN BY PCR METHOD

The foodborne pathogens cause serious public health problems in each country. In this regard, microbiological investigation is included in food safety management of the food chain. Molecular methods and mostly polymerase chain reaction (PCR) are considered highly sensitive, specific and rapid methods for pathogens detection from raw material and food. This study describes the using of specially designed and highly specific primers for PCR to identify 5 common and especially dangerous causeve agents of food poisoning and disease and to determine their level of distribution in food of animal and plant origin. The studies included the identification of methicillin-resistant Staphylococcus aureus (MRSA) and Cronobacter spp. (E. sakazakii) from raw milk, Shiga toxin-producing strains of Escherichia coli (STEC) from beef and swine carcasses, Bacillus cereus and Clostridium perfringens from various types of plant and animal raw materials and products of its processing - fruits, vegetables, berries, dried and preserved products, food concentrates, half-canned food. A total of 397 food samples were investigated to detect these pathogens using classical bacteriological methods and PCR. It was found that the distribution of foodborne pathogens in the studied products of animal and plant origin was as follows: Staphylococcus aureus (MRSA) and Cronobacterspp. (E. sakazakii) in raw cow milk in 6.5% and 19.4% of cases, respectively; shiga-toxin-producing Escherichia coli (STEC) from beef and pork carcasses in 8.1% and 5.7%; Bacillus cereus and Clostridium perfringens from different types of plant and animal raw materials and their processing products averages 27.5 % and 7.7 %, respectively. The advantages of molecular biological methods to which the PCR method relates, include their speed, as well as the specificity of identification of microorganisms by the features of genetic regions of genes that carry information about their pathogenicity factors. It has been found that the rate of detection of these pathogens when using the PCR method in comparison with classical methods increases at least 5-9 times. This data will be useful for assessing microbiological risk and will help authorities develop strategies to reduce consumer health risks.

HLA Polymorphisms and Haplotype Diversity in Transylvania, Romania.

Transylvania is a historical region in the northwestern part of Romanian with a rather heterogeneous population. Our study is the first to determine human leukocyte antigen (HLA) profiles in a large population sample from this region and to compare them with other European population groups. HLA genes were examined in 2,794 individuals using the Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) methods. All samples were tested for the HLA-A locus, 2,773 for HLA-B, 1,847 for HLA-C, and 2,719 for HLA-DRB1 loci. HLA gene frequency data from several European population groups (as presented in studies involving more than 1,000 individuals) served as reference in comparison with the local sample. The distribution of HLA genes in the studied population group was heterogeneous, as the Hardy-Weinberg equilibrium was statistically significant (P value < 0.01). The most common genes found in our sample group were A∗02 (0.27%), B∗35 (0.14%), C∗07 (0.25%), and DRB1∗11 (0.19%). The most common haplotype was A∗01~B∗08~C∗07~DRB1∗03 (1.26% in 1,770 individuals with complete data). This analysis confirmed the known heterogeneity of the Transylvanian population. The study indicates that the European population groups located in close vicinity (those from Serbia, Hungary, Wallachia, and Croatia) are genetically closest to the Transylvanian population.

Simple and accurate visual detection of single nucleotide polymorphism based on colloidal gold nucleic acid strip biosensor and primer-specific PCR

Single nucleotide polymorphism (SNP) was associated with many human diseases, therefore, SNP detection was important for early diagnosis and clinical prognosis. Herein, a simple and accurate method for visual detection SNP sites (A/A, G/G, A/G) in CYP1A1 gene related to cancers based on colloidal gold nucleic acid strip biosensor and primer-specific polymerase chain reaction (PCR) was established. This method could directly distinguish SNP sites on strip biosensor by introducing twice PCR amplifications. The second PCR (primer-specific PCR) was performed using specific product of the first PCR as template, thus this twice PCR could reduce non-specific amplification greatly and obtain target product. In addition, single-strand or double-strand DNA (ssDNA or dsDNA) was accurately produced by introducing mismatched base at the 3’ end of forward primers in primer-specific PCR. The designed strip biosensor could only combine with the ssDNA, thus visual detection of SNP could be achieved within 10 min by color difference of a pair of strips. 61 human blood samples by this method were identical with those of pyrosequencing. This method had the advantages of rapid, visual and low-cost and was expected to be applied in medical diagnosis.

Association of STAT3 gene polymorphism with susceptibility to asthma in children

Objective To study the relationship between STAT3 gene (rs2293152, rs957970) polymorphism and susceptibility to bronchial asthma in children, and to lay the foundation for search of the candidate pathogenic gene of asthma and early prevention of the disease. Methods One hundred and thirty hospitalized asthmatic children or out-patients with asthma at Affiliated Hospital of Southwest Medical University from September 2013 to September 2017 were selected as asthmatic group, and at the same time 145 healthy children were selected from the Affiliated Hospital of Southwest Medical University as the healthy control group.Apply sequence specific primers-polymerase chain reaction (SSP-PCR) was applied to detect susceptibility single nucleotide polymorphism (SNP) of STAT3 gene (rs2293152, rs9579700). The statistical differences in distribution between genotype and allele were analyzed between the asthma group and the healthy control group. Results The distribution frequency of CC genotype of rs2293152 in STAT3 gene was significantly higher in the asthmatic group (19.2%)than that in the healthy control group (9.0%) (χ2=6.065, P=0.014). C allele was significantly higher in asthmatic group (41.9% vs.33.8%) (χ2=3.861, P=0.049). There was no significant difference in the distribution of rs957970 in STAT3 gene between the asthma group and the healthy control group. Conclusions The SNP of rs2293152 in STAT3 gene is associated with susceptibility to asthma in children in South Sichuan.C allele may be the susceptible gene for childhood asthma, while the SNP of rs957970 in STAT3 gene has no significant correlation with childhood asthma in Southern Sichuan. Key words: STAT3 gene; Single nucleotide polymorphism; Asthma; Susceptibility; Child

CD44 Gene rs8193 C Allele Is Significantly Enriched in Gastric Cancer Patients.

Objective Gastric cancer is a multifactorial disease. In addition to environmental factors, many genes are involved in this malignancy. One of the genes associated with gastric cancer is CD44 gene and its polymorphisms. CD44 gene plays role in regulating cell survival, growth and mobility. The single nucleotide polymorphism (SNP) rs8193, located in the CD44 gene, has not been studied in gastric cancer patients of the Iranian population. The present study aims to study this polymorphism in 86 gastric cancer patients and 96 healthy individuals. Materials and Methods In this cross-sectional case-control study, rs8193 polymorphism was genotyped by allele specific primer polymerase chain reaction (ASP-PCR) technique. The obtained data were statistically analyzed. To find the potential mechanism of action, rs8193 was bioinformatically investigated. Results rs8193 C allele played a risk factor role for gastric cancer. Patients carrying this allele were more susceptible to have gastric cancer, with lymph node spread. On the other hand, rs8193 T allele, a protective factor, was associated with a higher chance of accumulation in the lower stages of cancer. C allele might impose its effect via destabilizing CD44 and miR-570 interaction. Conclusionrs8193 is statistically associated with the risk of malignancy, lymph node spread and stage of gastric cancer in Iranian population.

Susceptibility to ERAP1 gene single nucleotide polymorphism modulates the inflammatory cytokine setting in ankylosing spondylitis.

To evaluate the association of ERAP1 gene single nucleotide polymorphisms (SNPs) with the risk of ankylosing spondylitis (AS) and their role in modulation of the inflammatory interleukin (IL)-17/IL-23 axis in the disease. For genotyping, 190 AS cases and 190 healthy controls were enrolled. After DNA extraction, all the subjects were genotyped for rs17482078, rs469876, and rs27038 polymorphisms using single specific primer polymerase chain reaction (PCR) assay. After isolation of peripheral blood mononuclear cells, RNA extraction and complementary DNA synthesis, real-time PCR using SYBR Green master mix was employed to determine messenger RNA (mRNA) expression of IL-17A and IL-23 in PBMCs. Using enzyme-linked immunosorbent assay, the concentration of these cytokines was determined in serum samples. It was observed that the A allele of rs27038 polymorphism significantly increased AS risk (odds ratio [OR] = 1.53, 95% CI =1.11-2.12; P = 0.0096). Moreover, AA and AG genotypes of this SNP were associated with increased (OR = 2.89, 95% CI = 1.42-5.85; P=0.0031) and decreased (OR = 0.57, 95% CI = 0.36-0.92; P=0.021), respectively, risk of the disease. The rs27038 SNP was associated with C-reactive protein level. There were significantly increased mRNA and serum concentrations of both IL-17A and IL-23 in AS patients compared with controls. Furthermore, AS patients with the AA in comparison to other genotypes for rs27038 SNP indicated significantly increased mRNA and serum concentration levels for both cytokines. This study demonstrated the association of ERAP1 gene rs27038 polymorphism with the risk of AS in an Iranian population. Additionally, it seems that rs27038 is involved in the modulation of the inflammatory IL-17/IL-23 axis in AS.

HLA-C*18:01 and KIR2DL2+C1 genetic variants are associated with low viral load in cART naïve HIV-infected adult Zimbabweans.

Polymorphisms in killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) gene families are implicated in differential outcomes of HIV infection. However, research findings on the influence of KIR and HLA-C polymorphism on HIV disease progression remain inconclusive. We thus investigated the association of KIR and HLA-C gene polymorphisms with plasma HIV load (VL) and CD4+ T lymphocyte (CD4) count in 183 chronically HIV-infected, combination antiretroviral therapy (cART) naïve Zimbabweans of Bantu origin. The presence or absence of 15 KIR genes were determined using sequence specific primer polymerase chain reaction while HLA-C typing was performed using chain termination DNA sequencing. Plasma VL was determined using the Cavidi Exavir viral load version 3 assay while CD4+ T lymphocytes were enumerated using flow cytometry. VLs and CD4 counts were compared between gene/genotype carriers and non-carriers using Mann-Whitney ranksum test. HLA-C*18:01 allele carriers had a significantly lower median log10 VL (2.87copies/mL [IQR;2.3-3.2]) than the non-C*18:01 carriers (3.33copies/mL [IQR; 2.74-3.9]), p = 0.018. Further, median log10 VL was significantly lower in KIR2DL2+C1 carriers (2.745 [IQR; 2.590-2.745]) than non-KIR2DL2+C1 carriers (3.4 [IQR; 2.746-3.412]), p = 0.041. Comparison of CD4 + T lymphocyte counts between C*08:02 allele carriers and non-C*08:02 carriers showed a significantly higher median CD4 count in C*08:02 carriers (548cells/µL [IQR;410-684]) than in non-carriers (428cells/µL [IQR;388-537]), p = 0.034. We conclude that the HLA-C*18:01 and KIR2DL2+C1 genetic variants are associated with low VL while the C*08:02 is associated with high CD4+ T lymphocyte count among cART naïve Zimbabwean adults with chronic HIV infection.

Comparative study for diagnosis of babesiosis and theileriosis in different age groups of cattle in some localities in Egypt with treatment trials

Babesia bovis, Babesia bigemina, Theileria annulata are the most important endemic blood parasites in Ismailia and Sharkia Provinces, Egypt, which transmitted by ticks causing babesiosis and theileriosis which characterized by fever, enlarged lymph nodes, pale mucous membranes and may lead to death. Therefore, the aim of our study is to determine the infection rates of B. bovis, B. bigemina and T. annulata in relation to age. We found that the rates by staining method were as the following: 12.90%, 6.45%, 38.71% and 4.84% for B. bovis, B. bigemina, T. annulata and mixed infection, respectively, while by polymerase chain reaction (PCR) the rates were 35%, 20%, 60% for B. bovis, B. bigemina and T. annulata, respectively. This study confirmed the incidence of infection with Babesia and Theileria species in relation to age group of animals was 77.78% in adult age group, 66.67% in less than 3 months age group and 53.19% in 3-6 months age group. Also, our study proved that the use of imidocarb and buparvoqune beside acaricides considered a good choice for treatment. Although our results proved that using of specific primers in PCR was a rapid and an accurate method for differential diagnosis between Babesia and Theileria species in cattle, we found that the use of staining method was more helpful especially in mixed infection cases. In the end, we recommend using both methods together to get better results.

Interleukin-6-174 promoter polymorphism and susceptibility to hepatitis B virus infection in Javanese individuals

Hepatitis B virus infection is a significant risk factor for cirrhosis and hepatocellular carcinoma. Interleukin-6, a cytokine with broad pleiotropic actions, plays important role in cellular and humoral immune responses to viral infection. The -174 G/C polymorphisms of interleukin-6 is a frequent polymorphism and may determine the outcome of viral infection. To find out the genetic association between the -174 G/C polymorphism and susceptibility to hepatitis B virus infection in Javanese individuals, blood samples from Javanese hepatitis B virus-infected individuals and from Javanese healthy individuals confirmed by serological and molecular assays were evaluated. Genomic DNA was extracted from peripheral blood and the sequence specific primer-polymerase chain reaction method was applied for genotyping. The frequencies of genotypes C/C, C/G and G/G in hepatitis B virus-infected samples were 5.3%, 31.6%, 63.1% and in controls were 20%, 38%, and 42%, respectively. The frequencies of G allele in hepatitis B virus-infected samples was higher than that of healthy samples (OR 2.4, 95%CI: 1.289-4.459, p = 0.0057). These findings suggest that the G allele was associated with susceptibility to hepatitis B virus infection in Javanese individuals.

SRNAPrimerDB: comprehensive primer design and search web service for small non-coding RNAs.

Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531306 and 2941669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. Supplementary data are available at Bioinformatics online.