Singleplex Polymerase Chain Reaction Research Articles

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Low-cost conventional PCR techniques enable simultaneous detection of bacterial sexually transmitted infections with enhanced sensitivity and specificity

PurposeNeisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Mycoplasma hominis (MH), the three most common treatable bacterial sexually transmitted infections (STIs) worldwide can lead to many complications if remain untreated. Screening of high-risk population with highly sensitive methods will lead to significant improvement in patient outcomes and will prevent downward transmission. The advantages of Polymerase chain reaction (PCR) based assay are not only high sensitivity and specificity, but also detection of multiple organisms in a single reaction which reduce the result turn-around time. The aim of the present study was to evaluate the feasibility of a multiplex PCR assay method targeting 16S rRNA gene for simultaneous detection of NG, CT and MH infection along with their trend and occurrence among high-risk population in Assam, Northeast India. MethodsA cross-sectional study was undertaken, where a total of 200 randomly selected patients from high-risk population were included. After validation of singleplex PCR, Multiplex PCR (M-PCR) was performed along with the traditional culture method for NG. Results & conclusionThe overall agreement of M-PCR with singleplex PCR was very high (100%). The occurrence of STI was found to be very high (101/200; 50.5%). Furthermore, co-infection was detected in 10/200; 5%) individuals. Infection was more common among young individuals (p < 0.05) and males out-numbered females (p < 0.05). The most common organism detected was CT (42/200; 21%) followed by NG (41/200; 20.5%) and MH (20/200; 10%). The M-PCR assay workflow is simple, cost effective and can be used in routine diagnostic laboratories with basic molecular facilities.

Molecular detection and antibiogram of Shiga toxin-producing Escherichia coli (STEC) from raw milk in and around Bahir Dar town dairy farms, Ethiopia

Illnesses associated with consuming infected milk and milk products are a widespread problem in low and middle-income countries. Shiga toxin-producing Escherichia coli (STEC) is a bacterium commonly found in raw milk and causes foodborne diseases ranging from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome. This study aimed to investigate the virulence gene and antimicrobial resistance profiles of Shiga toxin-producing E. coli strains isolated from raw milk in dairy farms in and around Bahir Dar town. Raw milk samples (n = 128) collected from December 2021 to July 2022 were cultured, and E. coli strains were isolated using standard methods. Shiga toxin-producing E. coli strains were identified genotypically by the presence of the virulence markers using a single-plex polymerase chain reaction. The antibiotic susceptibility testing of Shiga toxin-producing E. coli isolates was done by the agar disk diffusion method. In total, 32 E. coli isolates were recovered from milk samples from lactating animals. PCR screening of these isolates resulted in 19 (59.3%) positives for Shiga toxin-producing E. coli. The stx2 gene was detected in 53% of cases, followed by stx1 (31%) and eae (16%. The STEC isolates were highly sensitive to ciprofloxacin (94.7%) and kanamycin (89.5%), while exhibiting significant resistance to amoxicillin (89.5%) and streptomycin (73.7%). The present study points out the occurrence of virulent and antibiotic-resistant Shiga toxin-producing E. coli strains in raw milk that could pose a potential risk to public health. Further analysis by whole genome sequencing is necessary for an in-depth assessment and understanding of their virulence and resistance factors. Moreover, large-scale studies are needed to identify the prevalence and potential risk factors and to prevent the spread of antibiotic-resistant STEC strains in the milk production chain.

Relationship, association and distribution of Helicobacter pylori infection in endoscopy patients at a Tertiary Hospital in Makurdi, Benue State, Nigeria to the symptoms of nausea and vomiting

The distribution of H. pylori obtained from patients referred for endoscopy at Benue State University Teaching Hospital (BSUTH) Makurdi, Benue State, Nigeria in relation to nausea and vomiting was examined in this study. To isolate H. pylori, gastric biopsy samples were obtained from the antrum of 80 consenting patients, and questionnaires were administered to them. The biopsies were placed in Brain Heart Infusion (BHI) broth and were used for isolation. Genomic Deoxyribonucleic Acid (gDNA) was extracted from the biopsies using ReliaPrep spin column method and used for singleplex Polymerase Chain Reaction (PCR). Out of the 80 patients, 24 (30%) had H. pylori infection as detected by 16S rRNA PCR. There was no significant association between infection and nausea/vomiting (OR = 5.622, 95% CI: 0.683-46.274; p = 0.076), or infection and hematemesis, common symptoms reported by patients (OR = 0.911, 95% CI: 0.304 - 2.729; p = 0.870). The research sheds light on the infection, prevalence and distribution of H. pylori in endoscopy referral patients at BSUTH, with respect to nausea and vomiting, and provides valuable information for both clinicians and researchers.

Molecular detection of Entamoeba gingivalis among periodontitis and gingivitis patients by singleplex polymerase chain reaction

Abstract Background: Periodontal disease is classically characterized by progressive destruction of the soft and hard tissues of the periodontal complex, mediated by an interplay between dysbiotic microbial communities and aberrant immune responses within gingival and periodontal tissues. It is being recorded as public health problems. Objective: The aim of study is to detect Entamoeba gingivalis parasite in the oral cavity of patients with periodontitis and gingivitis by using molecular technique with singleplex polymerase chain reaction (PCR). Materials and Methods: A total of 100 patients with periodontal diseases (periodontitis and gingivitis) were enrolled in the current study. Samples of dental plaque were collected from each patient and stained with Giemsa stain and studied under a microscope. Six samples were selected to be examined by using singleplex PCR technique for the detection of 18S-the small subunit of ribosomal RNA gene (SSU rDNA) gene with 203 bp and comparing the results. Results: This result showed a high significant prevalence of E. gingivalis in dental plaque samples in patients with periodontitis and gingivitis. About 46 (46%) samples were positive by microscope examination. In comparison, six positive samples (3 men and 3 women) were selected for parasitic investigation by singleplex PCR and the results of PCR with specific primers designed for the detection of 18S SSU rDNA gene of (203 bp) showed positive results for the six samples. Conclusion: It was concluded that E. gingivalis had a prevalence among male patients than female patients. In addition, singleplex PCR is the technique of choice for the detection of the target sequence of DNA.

Performance of microscopy compared to conventional PCR in identification of soil-transmitted helminth infections among antenatal women in a low-prevalence setting

PurposeTraditional microscopy-based methods may provide inaccurate estimates of Soil transmitted helminth (STH) infections in mild intensity of infection. Therefore, we aimed to determine the prevalence of STH infections using molecular diagnostic methods and compare the diagnostic performance of microscopy with polymerase chain reaction (PCR) in stool samples collected from pregnant women in primary care settings in Puducherry, India. MethodologyA singleplex PCR assay was developed to detect three species of STHs, namely Ascaris lumbricoides, Necator americanus, and Ancylostoma duodenale, by targeting the internal transcribed spacer regions (ITS1 and ITS2) of 5.8S rRNA. The PCR generated 420, 662, and 515 base pairs of DNA for the respective organisms. In addition to singleplex PCR, wet and concentration microscopy techniques were used. The results were expressed as percentages with 95% confidence intervals, and the diagnostic performance of microscopy was compared with PCR in terms of sensitivity, specificity, and positive, negative predictive values and kappa statistics. ResultsAmong the 650 pregnant women included, 48.8% were aged 25 years or less, 59% were primigravida, and half were from rural areas. The overall prevalence of any STH infection was higher in PCR compared to microscopy (8.9% vs. 7.2%). The prevalence of Ascaris lumbricoides was higher by microscopy (5.4% vs 2.6%), while the prevalence of Necator americanus was higher by PCR (6.3%) than by microscopy (1.8%). No species of Ancylostoma duodenale was detected. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of microscopy for detecting any STH infection was 22.4%, 94.3%, 27.7%, and 92.5%, respectively. The agreement between microscopy and PCR for the identification is as follows: for any STH infection, k ​= ​0.12, Ascaris k ​= ​0.16, and Necator k ​= ​0.20, respectively. ConclusionThe prevalence of any STH infection identified by PCR was higher than microscopy, and the agreement between the two methods was poor.

Development of a Multiplex Polymerase Chain Reaction (PCR) Assay for the Potential Detection of Insect Contaminants in Food

Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them targeting the amplification of two regions of the insect’s mitochondrial cytochrome c oxidase subunit I (COI-Fa and COI-Fb) and the other targeting a region of the nuclear protein-coding wingless (wg) gene. Using singleplex and multiplex polymerase chain reaction (PCR), we evaluated the three sets of primers using genomic DNA (gDNA) from 48 insect species including food storage insect pests and known vectors of foodborne pathogens. Seven plant-based food matrices were also evaluated for exclusivity testing. Additionally, we spiked fragments from five insect species in a selected food matrix (whole wheat flour). Singleplex and multiplex PCR amplified single specific bands (401–449 bp), corresponding to the wg gene, from insect species belonging to families Blattidae and Formicidae, and in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The COI-Fa primers amplified specific bands (171–188 bp) in all Dipteran species and the COI-Fb primers amplified a specific band (∼140 bp) in DNA from Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and P. interpunctella. However, the presence of specific bands in most Coleopterans was not consistent. No amplicon bands were observed in any of the food matrixes tested and the expected pattern of amplicon bands was seen in multiplex reactions using gDNA from spiked food samples. Our multiplex PCR assay targeted specific groups of insects that commonly contaminate foods without amplifying bands from the food matrixes tested; thus, molecular methods may be suitable for detecting insects or their fragments in foods.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Variable Virulence Genes in Clinical Isolates of Burkholderia pseudomallei : Impact on Disease Severity and Outcome in Melioidosis.

Objectives To isolate Burkholderia pseudomallei from clinical specimens and study the association of virulence genes with clinical manifestations and outcome in patients with melioidosis. Materials and Methods Burkholderia pseudomallei isolates obtained from melioidosis cases diagnosed during 2018 to 2021 were identified using VITEK 2 system and confirmed by polymerase chain reaction (PCR) targeting a Type III secretion system gene cluster. Multiplex PCR was performed to detect the genotypes of lipopolysaccharide (LPS) namely A, B, and B2, and singleplex PCR was performed to detect the presence of the Burkholderia intracellular motility gene ( BimA ) and filamentous hemagglutinin gene ( fhaB3 ). Statistical Analysis Chi-square/Fisher's exact tests were performed to study the association between various clinical manifestations and outcome and different virulence genes. The results were expressed as unadjusted odds ratios with 95% confidence intervals. Results Sixty-seven isolates were available for characterization. BimA Bm and BimA Bp were observed among 82 and 18% of the isolates, respectively. Both sepsis and mortality were significantly associated with BimA Bm . Majority of the isolates had fhaB3 (97%). Most of the isolates showed the presence of LPS A gene (65.7%) followed by LPS B gene (6%), while LPS B2 was not detected. Nineteen isolates could not be assigned to any LPS genotypes. Conclusion Among the virulence genes studied, only BimA Bm was significantly associated with sepsis and mortality. More than a quarter (28.3%) of the isolates could not be assigned to any LPS genotypes, hinting at a greater genetic diversity in our isolates.

Seasonal Prevalence and Molecular Identification of Thermophilic Campylobacter from Chicken, Cattle, and Respective Drinking Water in Kajiado County, Kenya.

Thermophilic Campylobacter species are a leading cause of human gastroenteritis throughout the world and have been implicated in reproductive disorders (abortion), mastitis, enteritis, and/or diarrhoea in livestock. A cross-sectional survey was conducted in Kajiado County to determine prevalence, seasonality, and molecular detection of thermophilic Campylobacter species (with emphasis on C. jejuni, C. coli, and other thermophilic Campylobacter species) in chicken, cattle, and respective pooled drinking water. A total of 457 samples comprising 265 cattle rectal swabs, 142 chicken cloacal swabs, and 50 trough water samples were collected from 55 randomly selected smallholder farms. Individual samples were subjected to standard techniques for isolation and biochemical tests, followed by singleplex polymerase chain reaction (sPCR) assays for identification and confirmation of genus and species. Overall, thermophilic Campylobacter prevalence was 35.4% (95% confidence interval (95% CI) = 31.0–39.8), with C. jejuni dominating at 55.6% (95% CI = 47.9–63.3%) over C. coli in all sample types. The highest thermophilic Campylobacter prevalence was observed in cloacal swabs of live chicken at 44.4% (95% CI = 36.2–52.6%), followed by rectal swabs from live cattle at 30.9% (95% CI = 25.3–36.5%). Water samples from cattle drinkers/trough were found to be contaminated at 34% (95% CI = 20.9–47.1%). The isolation rate was higher in cattle under the confinement system (44.3%) (95% CI = 36.1–52.5%) than in those under the free-roaming grazing system. Thermophilic Campylobacter species were isolated in both seasons, with higher prevalence (39.8% (95% CI = 33.6–45.9)) recorded during rainy and cold season in all sample types except for water. There was significant (P < 0.05) association between season and thermophilic Campylobacter occurrence, even though there were no statistical differences in the prevalence values across the two seasons. Results of this study demonstrate that cattle, chicken, and respective drinking water harbour potentially pathogenic thermophilic campylobacters, with C. jejuni being widely distributed among farms. It is possible that seasonal variations and cattle confinement result in differences in thermophilic Campylobacter carriage. Further epidemiological and phylogenetic studies comparing distribution of thermophilic Campylobacter spp. isolates in livestock, environmental, and human samples are recommended to establish source attribution to reduce the impact of resultant diseases for the wellbeing of public and livestock.

The Utility of Nonroutine Intraocular Fluid Polymerase Chain Reaction for Uveitis in Indonesia.

PurposeTo investigate the utility of nonroutine polymerase chain reaction analysis of intraocular fluid to guide the diagnosis of infectious uveitis.Patients and MethodsA retrospective cohort study was conducted by reviewing medical record data from intraocular fluid samples of uveitis patients who underwent single-plex real-time polymerase chain reaction analysis at the Department of Ophthalmology, Faculty of Medicine, Universitas Indonesia – Cipto Mangunkusumo Kirana Eye Hospital between January 2014 and December 2018.ResultsThe positivity rate of nonroutine polymerase chain reaction analysis was 17.2%. The vitreous sample tended to show a higher positive outcome (28.6%) than the aqueous sample (16.2%), even though the outcome was not statistically significant. Mycobacterium tuberculosis and Toxoplasma gondii were the most frequently observed microorganisms in the polymerase chain reaction analysis among uveitis patients in our setting. The duration of symptoms, type of sample fluid (aqueous/vitreous), or presence of anterior chamber cells ≥2 were not significantly associated with polymerase chain reaction positivity (p > 0.05).ConclusionNonroutine polymerase chain reaction analysis of intraocular fluid among a cohort of Indonesian patients demonstrated low positivity. The sensitivity and specificity of nonroutine single-plex polymerase chain reaction could not be estimated due to limitations such as lost to follow-up patients and incomplete monitoring data. The use of multiplex polymerase chain reaction in the future may be beneficial in our setting.

Genotypic and phenotypic tetracycline-based properties of Trueperella pyogenes isolates from bovine samples.

The purpose of this study was to investigate the tetracycline resistance in Trueperella pyogenes isolates from bovine samples in Burdur, Turkiye, and assess 16 tetracycline-resistance genes distribution among the isolates. Forty-nine T. pyogenes isolates were phenotypically characterized for anti-microbial resistance to doxycycline, oxytetracycline and tetracycline by disc diffusion method. Presence of tetracycline genes of T. pyogenes was investigated by multiplex and singleplex polymerase chain reaction. Our results indicated that 87.80% and 42.86% of the isolates were resistant to tetracycline and oxytetracycline, respectively, and the rate of resistance to doxycycline was 6.12%. Total of 21 (42.85%) were carrying tetracycline-resistance genes and tet(A) was present in 12 (24.49%) isolates; whereas, the tet(W) gene was identified in 9 (18.37%) and 2 (4.08%) of the isolates carried both tet(A) and tet(W), respectively. The study indicated antibiotic resistance patterns of tetracycline agents and links to the tet-genes among T. pyogenes were detected. It makes it worthwhile that this is the first report for detection of tet(A) gene in T. pyogenes.

Etiology of Severe Pneumonia in Children in Alveolar Lavage Fluid Using a High-Throughput Gene Targeted Amplicon Sequencing Assay.

Objective: To evaluate the diagnostic value of a high-throughput gene targeted amplicon sequencing (TAS) assay for detecting pathogenic microorganisms in alveolar lavage fluid (ALF) from children with severe community-acquired pneumonia (SCAP).Methods: A retrospective study was performed on 48 frozen ALF samples from 47 severe pneumonia cases admitted to Children's Hospital of Fudan University from January 1, 2019, to March 31, 2019. All samples were tested by a multiplex PCR (Multi-PCR) assay and a TAS assay. The results of the TAS panels were parallel compared with Multi-PCR and Conventional Tests (CT) including culture, direct fluorescent antibody method (DFA), and singleplex polymerase chain reaction (PCR).Results: The proportion of pathogens detection by CT was 81.2% (39/48). The 8 common respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), influenza A virus (FLUA), influenza B virus (FLUB), parainfluenza virus 1–3 (PIV1-3), and human Metapneumovirus (hMPV) were found in 31.2% (15/48) of the 48 samples by DFA. With the criteria of CT results used as “Golden Standard” for determing of TAS results, the proportion of pathogens detection by TAS was 70.8% (34/48). The difference of proportion of pathogens detection between TAS and CT was not statistically significant (p = 0.232). The sensitivity and specificity of TAS for pathogens detection based on CT were 87.1% (95% CI, 71.77–95.18%) and 100.0% (95% CI, 62.88–100%), the positive predictive value (PPV) and negative predictive value (NPV) were 100.0% (95% CI, 87.35–100%) and 64.2% (95% CI, 35.62–86.02%), respectively. While Multi-PCR results were used as “Golden Standard,” the total pathogens detection rate of TAS was 83.3% (40/48), which had a significant difference with that of Multi-PCR (p = 0.003). The sensitivity and PPV of TAS compared with Multi-PCR were 83.3% (95% CI, 69.23–92.03%) and 100.0% (95% CI, 89.08–100%), respectively. High rates of co-infection were proved by CT, Multi-PCR, and TAS. Mycoplasma pneumoniae (MP) and ADV were the two most frequently detected pathogens in all three assays.Conclusion: Compared with the CT and Multi-PCR methods, this TAS assay had a good performance in detecting bacteriological and viral pathogens from ALF. More research is needed to establish interpretation criteria based on TAS reads or analysis platforms.

Performance of TaqMan probes for the detection of sexually transmitted infections in South African women.

Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis and Mycoplasma genitalium are the four main aetiologies of sexually transmitted infections responsible for vaginal discharge syndrome (VDS). Commercially available multiplex polymerase chain reaction (PCR) assays are expensive and generally not customisable. We evaluated a highly customisable singleplex PCR approach by testing it in parallel with the Anyplex™ II STI-7 detection assay in a cohort of South African women that presented with VDS between May 2016 and January 2017. Our multiple singleplex PCR strategy proved to be a simple, accurate, rapid, affordable and scalable option for diagnosing VDS.

UreC and ZapA virulence genes amplification in clinical specimen of Proteus mirabilis in Bayelsa state, Nigeria

Background and objective: Proteus mirabilis is part of the Enterobacteriaceae family, Gram negative bacterium which typically lives in the human gut, which means when it causes illness it can be a serious bloodstream infection, urinary tract infection or disseminated infection. UreC and ZapA virulent genes constitute the major pathogenicity of this organism as well as its resistance to antibiotics. This study was carried out to detect the presence of UreC and ZapA genes in Proteus mirabilis isolates sourced from Federal Medical Centre and Niger Delta University teaching hospital in Bayelsa State. Materials and methods: A total of one hundred and forty (140) clinical samples were collected from Federal Medical Centre (FMC) Yenagoa and Niger Delta University Teaching Hospital (NDUTH) Okolobiri, Bayelsa State. Of the 140 samples collected, 64(45.7%) were from males while 76(54.3%) were from females. The samples were Urine, Sputum, High vaginal swab, Urethral swab, Ear swab and Wound swab. The samples were inoculated in different laboratory media and incubated at 37°C for 48 hours. Morphological, cultural, biochemical characteristics and Polymerase Chain Reaction (PCR) technique were noted appropriately. Means and corresponding standard deviations were calculated for continuous data while proportions, along with the 95% confidence intervals, were calculated for categorical data. Results: A total of 81 bacterial isolates were obtained from these samples, of which 17(20.9%), 22(27.2%), 8(10.0%), 10(12.3%) and 24(29.6%) were Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus respectively. Fifteen (15) out of the 17(20.9%) P. mirabilis isolates were subjected to single-plex PCR amplification using specific primers after extraction of bacterial DNA from the samples. Out of the 15 samples, 14(93.3%) were positive for UreC gene while 15(100%) were positive to ZapA gene. Conclusion: The present study revealed that virulent genes-UreC and ZapA are highly present in P. mirabilis isolates obtained from clinical specimens from FMC and NDUTH in Bayelsa state, thus making them more pathogenic and resistant to antibiotics curing effect.

Detection of Aspergillus fumigatus by Polymerase Chain Reaction (PCR)

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

61. PREIMPLANTATION GENETIC TESTING FOR MONOGENIC DISEASE OF SPINAL MUSCULAR ATROPHY BY MULTIPLE DISPLACEMENT AMPLIFICATION: 11 UNAFFECTED LIVEBIRTHS

Introduction PGT-M has become an effective method for providing couples with the opportunity of a pregnancy with a baby free of spinal muscular atrophy (SMA). Multiple displacement amplification (MDA) overcome the innate dilemma of very limited genetic material available for PGT-M. This study mainly evaluated the use of MDA, combined with haplotype analysis and mutation amplification, in preimplantation genetic testing for monogenic disease (PGT-M) for families with SMA. Material and methods MDA was used to amplify the whole genome from single blastomeres or trophectoderm (TE) cells. Exon 7 of the survival motor neuron gene 1 (SMN1) and eleven STRs markers flanking the SMN1 gene were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products. Results Sixteen cycles (19 ovum pick-up cycles) of PGT-M were initiated in 13 patients. A total of 141 embryos were diagnosed: 90 embryos were biopsied at the cleavage stage and 51 embryos were biopsied at the blastocyst stage. MDA was successful on 94.44% (85/90) of the single blastomeres and on 92.16% (47/51) of the TE cells. And the PCR efficiency were 98.4%(561/570) and 100%(182/182), respectively. In addition, the average allele drop-out (ADO) rates were 13.3%(60/392) and 9.8% (11/112), respectively. The results for SMN1 exon 7 were all matched with haplotype analysis, which allowed an accurate diagnosis of 93.62% (132/141) embryos. Twelve families had unaffected embryos available for transfer and a total of 38 embryos were transferred in 20 embryo transfer cycles. Eight transfers were successful, resulting in an clinical pregnancy rate of 40%(8/20) and an implantation rate of 28.95%(11/38). Finally,11 healthy babies were born. Among them, 5 patients were singleton live births and and 3 patients had twin births. Conclusion Careful handling during the MDA procedure can improve subsequent PCR efficiency and reduce the ADO rate. We suggest that this protocol is reliable for increasing the accuracy of the PGT-M for SMA.

Multiple displacement amplification as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease for β-thalassemia.

To evaluate whether using multiple displacement amplification (MDA) as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease (PGT-M) for β-thalassemia. This is a retrospective cohort study. All included patients underwent PGT-M cycles (n = 307) for β-thalassemia in our center from January 2014 to February 2018. We divided the patients into two groups based on two different detection methods. For the polymerase chain reaction (PCR) group (n = 115), multiplex nested PCR+ reverse dot blot analysis was performed directly after cell lysis. For the MDA group (n = 192), the whole genomes of single cells were directly amplified using MDA and then examined by singleplex PCR + reverse dot blot for β-thalassemia. A total of 2315 embryos were tested. The overall diagnostic efficiency of the MDA group was significantly higher than that of the PCR group (96.99% vs 88.15%, P < 0.001). The percentage of embryos available for transfer was significantly higher in the MDA group than in the PCR group (74.28% vs 64.98%, P < 0.001). Furthermore, the carrier embryo rate of the MDA group was significantly higher than that of the PCR group (50.11% vs 35.95%, P < 0.001). This study indicates that MDA, as the first step in PGT-M for β-thalassemia, can increase diagnostic efficiency.

Characterisation, verification and genetic basis of anomalous STR patterns: a report of four cases of X-chromosome STR biallelic patterns in human males.

Analysis of the characteristics and genetic basis of the anomalous short tandem repeat (STR) pattern encountered in forensic cases has been shown to be useful for analysing STR profiles in routine forensic casework. Here, we report biallelic patterns at several X-chromosome STR (X-STR) loci in human males revealed by forensic parameters investigation using the commercial AGCU X19 Kit. The presence of these patterns was verified by reanalysis using new samples and bidirectional Sanger sequencing of the singleplex polymerase chain reaction (PCR) products. And the genetic basis for their production was inferred based on the relative peak heights at the amelogenin locus and the affected locus (DXS10159, DXS10134 and DXS10079) and the normalised peak height ratios between the affected locus and adjacent loci relative to the control sample 9947A. The inference results suggested that two cases of biallelic pattern at the DXS10159 locus would be caused by local duplications, while in the other two cases, both the biallelic patterns at loci DXS10134 and DXS10079 would be due to somatic mutations. One case where the male showed a biallelic pattern at the DXS10159 locus (Xp11.21) was further analysed. Quantitative PCR (qPCR) revealed a microduplication (< 0.2Mb) spanning at least 13.9 kb in Xp11.21 encompassing the DXS10159 locus. Finally, a workflow for analysing anomalous STR patterns was summarised. In conclusion, this study is a detailed report of X-STR biallelic patterns in human males, which serves as an effective complement to the database and provides an example for the analysis of anomalous STR patterns.

Profile interpretation of extremely long alleles at DYF387S1 and DYS447 migrated into allele range of adjacent loci

Two rare cases of long alleles at Y-chromosome short tandem repeat (Y-STR) loci (DYF387S1 and DYS447) were identified when two father–son pairs were analyzed by multiplex amplification. “Null alleles” were observed at DYF387S1 and DYS447, and duplicated alleles were displayed at DYS533 and DYS19. We secondly amplified DYF387S1, DYS533, DYS447, and DYS19 loci by singleplex polymerase chain reaction (PCR) and sequence analysis of the long alleles at DYF387S1 and DYS447 loci. The results showed that alleles from DYF387S1 (allele 55) and DYS447 (allele 41) were longer than their common sizes in the allelic ladder ranges (33–42 for DYF387S1 and 18–30 for DYS447) and located in the neighboring loci (DYS533 and DYS19, respectively). Therefore, to identify these cases involving this unusual phenomenon, not only re-amplification using the same kit but also additional amplification (using alternative multiplex kits with different adjoining markers or additional singleplex PCR amplification) should be performed to avoid misinterpreting Y-STR profiles.