Polymerase Chain Reaction Primers Research Articles

Bioinformatic Comparisons of Some Web-based PCR Primer Design Programs

Bioinformatics has become an indispensable tool for both basic and applied research in biotechnology in the life sciences. The polymerase chain reaction (PCR) is a laboratory method that can be used to quickly amplify a large number of identical copies of a specific DNA segment. In PCR, short synthetic DNA fragments known as primers are used to selectively amplify a specific section of the genome. For PCR to be as efficient and specific as possible, it is important to choose an effective primer sequence and use the correct concentration of primers. If the primer is not designed carefully, non-specific amplification and/or primer dimer formation may occur, which may prevent product formation. Currently, a number of different design tools are available on the internet to assist molecular geneticists in designing PCR primers under optimal conditions. In this study, out of 39 web-based PCR primer design programs, 7 accessible, freely available and widely used web-based PCR primer design programs (NCBI, Primer3, Biserach, Genscript and Primer3plus; Stitcher 2.0; and PrimerQest Tool) were compared using bioinformatics applications for genomic sequences. The advantages and disadvantages of the web-based PCR programs are discussed on the basis of the comparison results.

The Bisulphite-Sequencing PCR for the Bovine Gstp1, Gsta4, and Ache Gene Methylation Study

Abstract Modifying cytosine by adding a methyl group in CpG dinucleotides within DNA sequence has several functions, but acts primarily as an important regulatory mechanism of mammalian gene expression. This consequently affects the phenotypic characteristics of the organism. The methylation profiles can be altered as a consequence of various environmental influences, age, disease, or diet, and can serve as biomarkers of important production and health traits. In this regard, the methylation profile analysis techniques are important. In the present study, we proposed and tested specific oligonucleotide primers for the bisulphite-sequencing polymerase chain reaction (BSP) analysis of the bovine GSTP1, GSTA4, and AChE genes, to optimize this approach for future studies of the methylation status of these genes. BSP can be used, for example, to study the effect of different pesticides and their combinations on the methylation of these and other bovine genes.

Development of a rapid, culture-free, universal microbial identification system using internal transcribed spacer targeting primers.

The indiscriminate administration of broad-spectrum antibiotics is a primary contributor to the increasing prevalence of antibiotic resistance. Unfortunately, culture, the gold standard for bacterial identification is a time intensive process. Due to this extended diagnostic period, broad-spectrum antibiotics are generally prescribed to prevent poor outcomes. To overcome the deficits of culture-based methods, we have developed a rapid universal bacterial identification system. The platform utilizes a unique universal polymerase chain reaction (PCR) primer set that targets the internal transcribed spacer (ITS) regions between conserved bacterial genes, creating a distinguishable amplicon signature for every bacterial species. Bioinformatic simulation demonstrates that at least 45 commonly isolated pathogenic species can be uniquely identified using this approach. We experimentally confirmed these predictions on a representative set of pathogenic bacterial species. We also show that the system can determine the corresponding concentration of each pathogen. Finally, we validated performance in clinical urinary tract infection samples.

Quantitative real-time PCR assays for species-specific detection and quantification of Baltic Sea spring bloom dinoflagellates.

In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples.

Rapid generation of homozygous fluorescent knock-in human cells using CRISPR-Cas9 genome editing and validation by automated imaging and digital PCR screening.

We previously described a protocol for genome engineering of mammalian cultured cells with clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here we are updating this former protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high-efficiency electroporation of optimized CRISPR-Cas9 reagents, screening of single cell-derived clones by automated bright-field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital polymerase chain reaction (PCR) and automated data analysis. Compared with the original protocol, our current procedure (1) substantially increases the efficiency of tag integration, (2) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (3) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with digital PCR. The increased efficiency of the new procedure reduces the number of clones that need to be analyzed in-depth by more than tenfold and yields to more than 26% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 d to 10 d during the overall ~10 week procedure, allowing a single person to process up to five genes in parallel, assuming that validated reagents-for example, PCR primers, digital PCR assays and western blot antibodies-are available.

Quantitative detection of <i>Pythium porphyrae</i> and <i>Pythium chondricola</i> (Oomycota), the causative agents of red rot disease in <i>Pyropia farms</i> in China

Red rot disease is one of the notorious algal diseases that threaten the cultivation of <i>Pyropia</i> in China, and two <i>Pythium pathogens</i>, i.e., <i>Pythium porphyrae</i> and <i>P. chondricola</i>, have been reported as causative agents. To monitor the pathogens, a fluorescent quantitative polymerase chain reaction (PCR) method was developed to quantitatively detect their abundance. Using overlapping PCR and pathogen-specific primer pairs, two pathogen-specific fragments were concatenated to construct an internal standard plasmid, which was used for quantification. For zoospores of known numbers, the results showed that this method can detect as less as 100 and 10 zoospores mL<sup>-1</sup> in a 200 mL solution for <i>P. porphyrae</i> and <i>P. chondricola</i>, respectively. Using monthly collected seawater at 10 sites in Haizhou Bay, a typical aquaculture farm in China, a significantly higher temperature and a significantly lower salinity were determined in December 2021. <i>P. porphyrae</i> was determined to be more abundant than <i>P. chondricola</i>, though with similar temporal distribution patterns from December 2021 to February 2022. When a red rot disease occurred in December 2021, the two pathogens were significantly more abundant at two infected sub-sites than the uninfected sub-site within both seawater and sediment, though they were all significantly more enriched in sediment than in seawater. The present method provides the capability to quantify and compare the abundance of two pathogens and also has the potential to forecast the occurrence of red rot disease, which is of much significance in managing and controlling the disease.

Genetic spectrum features and diagnostic accuracy of four plasma biomarkers in 248 Chinese patients with frontotemporal dementia.

Frontotemporal dementia (FTD) is characterized by phenotypic and genetic heterogeneities. However, reports on the large Chinese FTD cohort are lacking. Two hundred forty-eight patients with FTD were enrolled. All patients and 2010 healthy controls underwent next generation sequencing. Plasma samples were analyzed for glial fibrillary acidic protein (GFAP), α-synuclein (α-syn), neurofilament light chain (NfL), and phosphorylated tau protein 181 (p-tau181). Gene sequencing identified 48 pathogenic or likely pathogenic mutations in a total of 19.4% of patients with FTD (48/248). The most common mutation was the C9orf72 dynamic mutation (5.2%, 13/248). Significantly increased levels of GFAP, α-syn, NfL, and p-tau181 were detected in patients compared to controls (all p<0.05). GFAP and α-syn presented better performance for diagnosing FTD. We investigated the characteristics of phenotypic and genetic spectrum in a large Chinese FTD cohort, and highlighted the utility of plasma biomarkers for diagnosing FTD. This study used a frontotemporal dementia (FTD) cohort with a large sample size in Asia to update and reveal the clinical and genetic spectrum, and explore the relationship between multiple plasma biomarkers and FTD phenotypes as well as genotypes. We found for the first time that the C9orf72 dynamic mutation frequency ranks first among all mutations, which broke the previous impression that it was rare in Asian patients. Notably, it was the first time the C9orf72 G4C2 repeat expansion had been identified via whole-genome sequencing data, and this was verified using triplet repeat primed polymerase chain reaction (TP-PCR). We analyzed the diagnostic accuracy of four plasma biomarkers (glial fibrillary acidic protein [GFAP], α-synuclein [α-syn], neurofilament light chain [NfL], and phosphorylated tau protein 181 [p-tau181]) at the same time, especially for α-syn being included in the FTD cohort for the first time, and found GFAP and α-syn had the highest diagnostic accuracy for FTD and its varied subtypes.

Comparative analysis of reverse-transcription-polymerase chain reaction for Aichivirus detection.

Aichivirus-A (AiV-A), a member of the Kobuvirus genus of the family Picornaviridae, was first reported in stool samples of patients with non-bacterial gastroenteritis in Aichi Prefecture, Japan, in 1989. AiV has been reported from in various aquatic environments, such as surface water and sewage, can be transmitted via the fecal-oral route through contaminated water. As AiV is known to acute gastroenteritis worldwide, developing methods for AiV detection from contaminated environments and food is required. In the present study, we established an effective polymerase chain reaction (PCR) method to detect AiV. Various real-time reverse transcription (RT)-PCR and conventional PCR methods for AiV detection were compared, and the limit of detection was confirmed by comparing the sensitivity at varied primer concentrations and PCR conditions. The final detection limits were 102 copy/μL in conventional PCR, and 101 copy/μL in the real-time RT-PCR. The optimized method used in this study might aid in detecting AiV contamination.

NEW PRIMER DESIGN of MULTIPLEX POLYMERASE CHAIN REACTION (PCR) for DETECTION of Escherichia Coli and Salmonella Enterica in FOOD SAMPLE

Background: Multiplex PCR techniques are used to detect multiple pathogens at the same time simultaneously. In order to achieve the specificity and sensitivity of detection, it is important to optimize the multiplex PCR method properly. Developing suitable primers and optimizing PCR temperature to boost particular genes from pathogens are key factors for this optimization. Methods: For the simultaneous detection of Escherichia coli and Salmonella enterica in food samples, this research aims to develop a set of primers for multiplex polymerase chain reaction (PCR). Using Primer-BLAST software, the study utilizes specific primer designs for the phoA gene (Eschericia coli) and invA gene (Salmonella enterica). DNA isolation has confirmed successful extraction from the two bacterial samples. PCR was performed under different conditions, including Singleplex and Multiplex PCR, using two annealing temperatures of 53oC and 50oC. Results: The results showed that this method can effectively amplify target genes and indicate their specificity and reliability at both temperature levels. Given these results, it has successfully conducted multiplex PCR using the built primer pairs. Both annealing temperatures of 50oC and 53oC can be used to perform multiplex PCR to detect E. coli and Salmonella enterica in one PCR reaction. Conclusion: Through this research, we have created a new set of Multiplex PCR Primers and an optimized multiplex PCR technique for the simultaneous detection of E coli and Salmonella enterica in food samples. The rapid and simultaneous screening of E. coli and S. enterica, which contributes to improved food safety measures and pathogen detection in the food industry, is promising with this optimized PCR approach.

QPrimerDB 2.0: an updated comprehensive gene-specific qPCR primer database for 1172 organisms.

High-quality primer design is essential for the success of all polymerase chain reaction (PCR)-based experiments. We previously developed a thermodynamics-based gene-specific quantitative PCR (qPCR) primer database for 147 organisms, which has been used extensively in gene expression studies. However, the number of organisms and the imperfection of function in the database limits its potential applications. Here, we improved the functionality of qPrimerDB to create a more comprehensive primer resource. Specifically, we (i) developed an improved primer design tool, qPrimer, building upon the previous qPrimerDB pipeline, to enhance the efficiency and simplicity of genome-scale qPCR primer design; (ii) pre-computed qPCR primer resources from 1 308 genomes of 1172 organismsand (iii) introduced a complete system for identifying, designing, checking, marking, and submitting qPCR primers. qPrimerDB 2.0 is freely available at https://qprimerdb.biodb.org. The qPrimer source code is available at https://github.com/swu1019lab/qPrimer.

Common RBC antigens in O type Tunisian blood donors and their importance in alloimmunization.

The presence of some red blood cell (RBC) antigens may affect the preference for using type O blood in emergency situations because they may induce complex or multiple alloimmunization in special circumstances. A subgroup of 77 type O blood Tunisian donors were genotyped for 19 common blood alleles using the single specific primer-polymerase chain reaction method. The statistical analysis was done using HaploView software. The study showed the dominance of the alleles RH*5, KEL*2, FY*2, and CO*1 and the absence of the homozygous state of the KEL*1 and CO*2 alleles. Furthermore, a complete linkage disequilibrium between the RH*2/RH*4 and RH*3/RH*5 loci and the FY*Null/FY*Exp and FY*A/FY*B loci was detected. Additionally, it seems that sensitization to MNS:3, FY:1, and RH:3 may constitute a potential factor for alloimmunization after transfusion with O blood type units: the probabilities of simple alloimmunizations are 24.5 per 100, 18.5 per 100, and 18 per 100, respectively. Multiple alloimmunization against RH:1;KEL:1 or RH:1;KEL:1;RH:3 phenotypes may occur, with probabilities of 7 per 1000 and 2 per 1000, respectively. Some O-type RBC units may contain blood with very immunogenic phenotypes, the use of which in an emergency requires great caution because it can be a step towards subsequent alloimmunization.

KIR2DL1 gene is a surrogate marker of protection against infection-related hospitalization among HIV-1 unexposed versus exposed uninfected infants in Cameroon

BackgroundHIV-exposed uninfected infants (HEU) appear more vulnerable to infections compared to their HIV-unexposed uninfected (HUU) peers, generally attributed to poor passive immunity acquired from the mother. This may be due to some genetic factors that could alter the immune system. We thus sought to determine the distribution of Killer Cell Immunoglobulin-Like Receptors (KIRs) genes in HEU versus HUU and study their associations with the occurrence of infection-related hospitalization.MethodsA cohort study was conducted from May 2019 to April 2020 among HEU and HUU infants, including their follow-up at weeks 6, 12, 24, and 48, in reference pediatric centers in Yaoundé-Cameroon. The infant HIV status and infections were determined. A total of 15 KIR genes were investigated using the sequence-specific primer polymerase chain reaction (PCR-SSP) method. The KIR genes that were significantly associated with HIV-1 status (HEU and HUU) were analyzed for an association with infection-related hospitalizations. This was only possible if, and to the extent that, infection-related hospitalizations varied significantly according to status. Multivariate logistic regression analyses were conducted to determine the association between KIR gene content variants and HIV status, while considering a number of potential confounding factors. Furthermore, the risk was quantified using relative risk, odds ratio, and a 95% confidence interval. The Fisher exact test was employed to compare the frequency of occurrences. A p-value of less than 0.05 was considered statistically significant.ResultsIn this cohort, a total of 66 infants participated, but only 19 acquired infections requiring hospitalizations (14.81%, 04/27 HUU and 38.46%, 15/39 HEU, p = 0.037). At week 48 (39 HEU and 27 HUU), the relative risk (RR) for infection-related hospitalizations was 2.42 (95% CI: 1.028–5.823) for HEU versus HUU with OR 3.59 (1.037–12.448). KIR2DL1 gene was significantly underrepresented in HEU versus HUU (OR = 0.183, 95%CI: 0.053–0.629; p = 0.003), and the absence of KIR2DL1 was significantly associated with infection-related hospitalization (p < 0.001; aOR = 0.063; 95%CI: 0.017–0.229).ConclusionCompared to HUU, the vulnerability of HEU is driven by KIR2DL1, indicating the protective role of this KIR against infection and hospitalizations.

Iraqi patients with a single-nucleotide polymorphism of interleukin-10 -1082G/A and interleukin-6 -174G/C susceptibility to asthma

The effects of genetic variations in the IL-10 -1082G/A gene and IL-6 -174G/C gene, as well as the genotypes and alleles linked to the prevalence of asthma disease, were investigated using a molecular and immunological study. Between October 2018 and the end of July 2020, 40 healthy individuals (20 females and 20 males) served as a control group for the study, which involved 50 asthmatic patients (31 females and 19 males) at the Allergy Centre, Al-Anbar Teaching Hospital, in Al-Anbar City. The study used the Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) technique to show that the single nucleotide polymorphisms IL-10 -1082G/A and IL-6 -174G/C had a considerably high prevalence rate (P&lt;0.05) among asthma case and that there was an association between the polymorphism and the asthma risk. The findings indicate that asthma patients had considerably higher (P&lt;0.05) IL-10 A alleles and heterozygous GA genotypes (1082G/A) compared to the control group. Genetic variations affecting IL-10 production and the genotypes affecting IL-10 serum levels are associated with the occurrence of asthma and are attributed to the IL-10 -1082G/A promoter gene polymorphism. There was a strong correlation between cytokine levels, of disease development, and the genotypes of the AA and AG genes, indicating that IL-10 -1082A/G predisposition to asthma may be influenced by the gene promoter polymorphism. Asthma development and immunological markers (IL-10) are substantially correlated. One theory links allergic rhinitis to both the development of asthma and its risk. Inducing long-term immunological and clinical tolerance in patients was a good use of HDM immunotherapy. The current study's findings indicate a substantial difference between the asthma patients and the control group in terms of gene type and allele frequency of the IL-6 -174G/C polymorphism. The patients exhibited a higher prevalence of the G allele and the GG homozygous genotype than the control group. Therefore, it was shown that those with GG genotypes had a 2-fold increased probability of having asthma, indicating that patients were more prone to the condition.

Human leukocyte antigen HLA-B*57:01 status in HIV-1 patients developing hypersensitivity reactions in Benin: a pilot study

BackgroundAntiretroviral drugs in people living with HIV-1 (PLHIV-1) often trigger side effects which may lead to discontinuation or failure of treatment. Human Leukocyte Antigen B*57:01 (HLA-B*57:01) allele is known to predict hypersensitivity reactions to Abacavir. Very few data are available on the prevalence of HLA-B*57:01 allele in PLHIV-1 in African countries. This study aimed to screen for HLA-B*57:01 allele in PLHIV-1 in Benin.MethodsThis pilot study was carried out on one hundred ten PLHIV-1 enrolled in two health facilities in Benin. Socio-demographic and clinical data were collected. Biological data were determined and HLA-B*57:01 allele was genotyped, using Single Specific Primer-Polymerase Chain Reaction in blood samples.Results70% of participants were female. PLHIV-1 were under TDF + 3TC + DTG (47.2%) or TDF + 3TC + EFV (57.3%). Their median age was 41 [36-48.75] years and the average CD4 + T cell count was 249 [130-381.25] cells/µl. The average viral load in treatment failure PLHIV-1 was 4.7 [3.9–5.2] Log10. At the inclusion date, twenty-nine (26.4%) PLHIV-1 under TDF + 3TC + EFV have developed hypersensitivity reactions. None of 110 patients had shown HLA-B*5701 allele.ConclusionOur study revealed that HLA-B*57:01 allele was very rare in PLHIV-1 in Benin, suggesting that its screening before starting the Abacavir regimen did not seem necessary.

Exploring the feasibility of utilizing universal primers in detecting mucormycosis pathogens: An in-silico analysis

This study aimed to design and evaluate a universal primer for Polymerase Chain Reaction (PCR)- based detection of mucormycosis-causing fungi by targeting their Internal Transcribed Spacer (ITS) sequences. In-silico analysis was conducted to assess primer suitability. Using Clustal Omega and Primer-BLAST, ITS sequences of 32 fungi species causing mucormycosis were aligned and subjected to primer design. Generated primers were sorted and in silico PCR simulations were performed to identify primers capable of amplifying all fungal species. Instead of identifying one pair of universal primer, in silico PCR analysis identified a panel of 14 primer pairs designed from full-length ITS sequences, and a panel of 12 primer pairs designed from conserved regions, that could detect 31 species. The study recommends a panel of 12 primers for multiplex-PCR to detect mucormycosis-causing fungi instead of a long list of PCR analyses for each fungus in diagnosing mucormycosis.

Development of Polymerase Chain Reaction-High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni.

Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire's Disease in humans. L. pneumophila is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR-high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.

Development of rapid and efficient enterovirus typing and classification platform for real-time diagnostics

Enteroviruses are highly prevalent among children and adults worldwide. Accurate detection and genotyping of enteroviruses can be achieved using consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction (PCR)-based methods. Nevertheless, the popularized use of such methods for enterovirus detection and genotyping is limited by their relatively long processing time (>2 days), high processing cost, and the requirement for specialized and expensive instrumentation. Here, we report a relatively simple asymmetric PCR-electrochemical biosensing method that can detect enteroviruses and accurately characterize them into four distinct genotypic subgroups in less than 2 h. Specifically, genes of interest in the enteroviruses were amplified via asymmetric PCR (asy-PCR) using the designed PCR primers. The asy-PCR products were hybridized with enterovirus genotype-specific single-stranded DNA primer probes. Changes in response to electrochemical impedance signals can rapidly clarify the genotypes of enteroviruses. We observed that this method could not only effectively amplify the target gene within minimal PCR-amplified cycles of consensus-degenerate hybrid oligonucleotide (COHOP) products from the specimen but also allowed the rapid differentiation of the four subgroups of enteroviruses in 2 h. In summary, we developed an efficient and rapid electrochemical biosensing platform for enterovirus subtyping diagnostics that can be applied to other emergent infectious diseases.

Association of HLA-DQ4/5 genotype polymorphisms with celiac disease in a group of children in Southwest Iran: A case-control study.

Celiac disease (CD) has proinflammatory and pathogenic immune responses to gluten in intestinal tissue, leading to structural changes in the mucosa of the small intestine. The association of human leukocyte antigen (HLA)-DQ2 and DQ8 genotypes with CD has been previously reported. This test has a negative predictive value close to 100%, so its main purpose is to rule out the detection of CD completely or almost completely. There is limited information regarding HLA-DQ4/5 in CD. This study was conducted to determine the HLA-DQ4/5 genotypes in a group of Southwestern Iranian children with CD. We conducted a case-control study in Southwest Iran involving 100 participants, employing a nonprobabilistic sampling method. Samples were taken from participants' oral buccal mucosa at Imam Sajjad Hospital of Yasuj, Iran. Then DNA was extracted from these samples and used to determine the frequency of HLA-DQ4/5 genotypes through Sequence-Specific Primer-Polymerase Chain Reaction assay. SPSS 20 was utilized for statistical analyses. Fifty diagnosed patients with CD (high anti-tissue transglutaminase [tTG]-IgA level [upper limit of normal] with pathological findings of Marsh III) and 50 non-CD individuals (normal anti-tTG-IgA level and normal total IgA level) were enrolled in the study from August 5, 2022 to October 15, 2023. Findings showed that the DQ4a*4b allele has the highest frequency in the CD samples (78%, p < 0.01) followed by the DQ5a*5b allele (12%, p < 0.01). Additionally, there was a higher prevalence of DQ4/DQ5 in patients with CD compared to controls (odds ratio = 6.5, confidence interval = 0.84 to 69.46, p < 0.01). Furthermore, a significant association was found among HLA DQ4/5 genotype, age (>9.5) (p < 0.01), and gender (female) (p < 0.05). The observed significant differences among HLA-DQ4 and HLA-DQ5 in Iranian CD samples against controls and the high value of the relative risks showed the significant function of the studied alleles in the prevalence of CD in Iranian patients.

Whole Exome Sequencing Indicating GGCCTG Hexanucleotide Repeat in Patients with Spinocerebellar Ataxia Type 36

Introduction: Spinocerebellar ataxia type 36 (SCA36) is caused by large GGCCTG repeat expansion in the NOP56 gene. The genetic diagnosis based on Southern blot is expensive and time-consuming. This study aimed to evaluate the reliability and effectiveness of whole exome sequencing (WES) for routine genetic diagnosis of suspected SCA36 patients. Methods: Pathogenic repeat expansions for SCAs including SCA36 were first analyzed based on WES data using ExpansionHunter in five probands from SCA families, then the results were confirmed by triplet repeat primed polymerase chain reaction (TP-PCR) and Southern blot. Results: GGCCTG repeat expansion in NOP56 was indicated in all five probands by WES, then it was found in 11 SCA patients and three asymptomatic individuals by TP-PCR. The sizes of GGCCTG repeat expansions were confirmed to be 1,390–1,556 by Southern blot. The mean age at onset of the patients was 51.0 ± 9.3 (ranging from 41 to 71), and they presented slowly progressive cerebellar ataxia, atrophy and fasciculation in tongue or limb muscles. Conclusion: The patients were clinically and genetically diagnosed as SCA36. This study proposed that WES could be a rapid, reliable, and cost-effective routine test for the preliminarily detection of SCA36 and other ataxia diseases.

Emergence of Plasmid-Mediated Quinolone Resistance (PMQR) Genes in Campylobacter coli in Tunisia and Detection of New Sequence Type ST13450.

The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in Campylobacter coli isolates collected from broilers, laying hens and poultry farm environments. One hundred and thirty-nine C. coli isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6)-Ib gene was performed using polymerase chain reaction (PCR) and specific primers. aac(6')-Ib amplicons were further analyzed by digestion with BtsCI to identify the aac(6')-Ib-cr variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 C. coli isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the qnrB, qnrS, qepA, and aac(6')-Ib-cr were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in C. coli isolates in Tunisia and globally.