PCR-based Techniques Research Articles

Detection of Toxoplasma Gondii among Women in Duhok City-kurdistan Region of Iraq

Background: Congenital toxoplasmosis is one of the most endemic parasitic infections that can lead to clinical complications by transmission through mother to fetus transplacentally in aborted women. Objectives: The aim of this study was to estimate the rate of toxoplasmosis among pregnant and aborted women in Duhok/ Kurdistan Region-Iraq by using Enzyme-Linked ImmunoSorbent Assay(ELISA) and detect the seropositive cases by Real-Time Polymerase Chain Reaction (RT-PCR) . Materials and Methods: A total of 102 samples were collected from women admitted to obstetrics and gynecology hospital in Duhok province. The data were collected through the period from January, 20th , 2019 until December, 30th, 2019.Blood and placental samples from 60 aborted women with Bad Obstetric History(BOH) and 42 placental samples from healthy normal pregnancies (as a control group). The placental samples of normal pregnancies were tested by RT-PCR technique while aborted women’s blood and placental samples were tested by both Enzyme-Linked ImmunoSorbent Assay(ELISA) and RT-PCR techniques. Results: The results revealed a low rate of infection by congenital toxoplasmosis. Seropositivity to T. gondii were 20/60 33.3% by ELISA, 25% IgM, 65% IgG and 10% both IgG and IgM whereas the retested seropositive samples by RT-PCR only 5% showed positive results. The highest rate of infection was at 32- 38 age group, first trimester 85%, single abortion 35%, who inhibits rural area 80%,ingesting homemade food 80%, and contact with animals(cats) 85%. Conclusion: Real-time PCR-based technique is the most accurate and suitable for routine screening, providing a rapid, sensitive, and quantitative way of detecting congenital toxoplasmosis in clinical specimens, without any false-positive result that may help to a targeted treatment.

Amplified Fragment Length Polymorphism: Applications and Recent Developments.

AFLP or amplified fragment length polymorphism is a PCR-based molecular technique that uses selective amplification of a subset of digested DNA fragments from any source to generate and compare unique fingerprints of genomes. It is more efficient in terms of time, economy, reproducibility, informativeness, resolution, and sensitivity, compared to other popular DNA markers. Besides, it requires very small quantities of DNA and no prior genome information. This technique is widely used in plants for taxonomy, genetic diversity, phylogenetic analysis, construction of high-resolution genetic maps, and positional cloning of genes, to determine relatedness among cultivars and varietal identity, etc. The review encompasses in detail the various applications of AFLP in plants and the major advantages and disadvantages. The review also considers various modifications of this technique and novel developments in detection of polymorphism. A wet-lab protocol is also provided.

Usefulness of matrix-assisted laser desorption ionization/time of flight mass spectrometry for the identification of Streptococcus mutans.

This study evaluated the reliability of MALDI-TOF MS coupled with statistical tools for the identification of Streptococcus mutans in comparison with PCR-based techniques. Bacterial isolates were identified and serotyped by conventional PCR, using S. mutans species and serotype-specific primers. For bacterial identification, mass spectra data from S. mutans and other streptococci were compared with Biotyper V 3.1 database and the mass peak lists were examined by cluster and principal component (PCA) analysis. Identification of potential biomarkers was performed using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and BLAST tool of the NCBI database. PCR identified 100% of the isolates as S. mutans. S. mutans strains were typed as serotypes c (85.6%), e (8.6%), k (4.8%), and f (0.9%). Although only the 70% of the strains tested were identified at species level by the Biotyper database, PCA and cluster analysis of mass peaks allowed the identification of 100% S. mutans isolates and its differentiation from the other oral and non-oral streptococci. One mass peak at m/z value of 9572.73 was identified as species-specific biomarker for S. mutans. No biomarkers were identified for S. mutans serotypes. KEY POINTS: • MALDI-TOF MS coupled with statistical tools for the identification of S. mutans. • Detection of species identifying biomarkers by MALDI-TOF MS. • PCR identification and serotyping of S. mutans from saliva samples.

The Sensitivity and Specificity of Novel Primers for Detection of BRAFV600E Mutation.

Objective:This study aimed to evaluate the sensitivity and specificity of a PCR-based novel technique for the detection of BRAF mutation in early stages of the cancer. Methods:Different lengths of primer sets, ranging from 8 bp to 20 bp, were designed and used in this study. These primers were developed by applying on cancer cell lines. After that, the sensitivity and specificity of the methodology was evaluated by making serial dilutions. Results:The quantitative allele specific discrimination PCR (QUASAqPCR) primer with 14 bp length was sensitive enough to detect significantly 1:1,000 ratio of BRAFV600E to wild-type background (P = 0.011), when using 150 nanograms of DNA from cell lines in the reactions. Conclusion:High sensitivity and specificity levels of QUASA-qPCR method can improve diagnostic accuracy for BRAF mutation testing in patients at early stages of cancers and help stratify the appropriate choice of treatment.

Prevalence and Epidemiology of Multidrug-Resistant Pathogens in the Food Chain and the Urban Environment in Northwestern Germany.

The surveillance of antimicrobial resistance among humans and food-producing animals is important to monitor the zoonotic transmission of multidrug-resistant bacteria (MDRB). We assessed the prevalence of four MDRB within the meat production chain, including extended-spectrum β-lactamase (ESBL)-producing, carbapenemase-producing Enterobacterales (CPE) and colistin-resistant Enterobacterales (Col-E), as well as vancomycin-resistant enterococci (VRE). In total, 505 samples from four stages of meat production, i.e., slaughterhouses, meat-processing plants, fresh food products and the urban environment, were collected in northwestern Germany in 2018/2019 and screened for the presence of MDRB using both culture-based and PCR-based techniques. We detected genes encoding for carbapenemases in 9–56% (blaOXA-48, blaKPC, blaNDM, blaVIM) and colistin resistance-encoding mcr genes in 9–26% of the samples from all stages. Culture-based analysis found CPE and VRE only in environmental samples (11% and 7%, respectively), but Col-E and ESBL-producers in 1–7% and 12–46% of samples from all stages, respectively. Overall, our results showed that ESBL-producers and mcr-carrying Col-E were common in food-producing animals at slaughterhouses, in meat-processing plants and in food items at retail, while CPE and VRE were only found in the environment. The discrepancy between detected carbapenemase genes and isolated CPE emphasizes the need for more sensitive detection methods for CPE monitoring.

Loop-mediated isothermal amplification: a rapid molecular technique for early diagnosis of Pseudomonas syringae pv. syringae of stone fruits

BackgroundPathogenic bacteria cause significant economic damages in agriculture. The detection of such bacteria is considered as a continual interest for plant pathologists to prevent disease dissemination. Pseudomonas syringae pv. syringae is one of the most important bacterial pathogens infecting yield and quality of stone fruits throughout the world. Biochemical assays such as a LOPAT and GATTa are common methods to detect this pathogen. Serological tests and culturing on King’s B selective medium also used to isolate this bacterium. Selective media is composed of specific and effective ingredients to inhibit the growth of certain species of microbes in a mixed culture while allowing others to grow. These are used for the growth of only selected microorganisms. King’s B medium can be used as a general medium for the non-selective isolation cultivation and pigment production of Pseudomonas species from foods, cosmetic samples, plants, etc.Nevertheless, the mentioned methods are not enough accurate to differentiate the strains. On the other hand, PCR-based techniques are sensitive and efficient in detecting plant diseases. However, these techniques are not practicable for those researchers who do not have access to a thermal cycler. We have used loop-mediated isothermal amplification to couple with a target. The amplification of syrD gene using loop and bumper primers can be used to prevent disease dissemination. ResultsThe outcome of this investigation indicated more sensitivity of LAMP in comparison to PCR. The direct addition of SYBR Gold in microtube is more sensitive than gel in both LAMP and PCR byproducts so we can eliminate gel electrophoresis, while the LAMP showed high sensitivity and high specificity in comparison to results obtained by cultivation. The described molecular test could detect Pseudomonas syringae pv. syringae type in nearly 1 h, and this is the first time that Lamp molecular detection of Pseudomonas syringae pv. syringae particularly on stone fruits is described and introduced. ConclusionsThe obtained data confirmed that LAMP is a fast, cheap, and high specific method for the rapid detection of Pseudomonas syringae pv. syringae to the comparison of PCR and culture.

Molecular Detection of Extended-Spectrum β-Lactamases- Producer Serratia marcescens Causing Neonatal Sepsis in Iraq

Serratia marcescens is an important nosocomial pathogen that causes a variety of infections, especially urinary tract and bloodstream infections. The emergence and spread of multidrug-resistant Serratia marcescens producing extended-spectrum beta-lactamases is a threat to public health worldwide at present. Extended-spectrum β-lactamases (ESβLs) including TEM, SHV, and CTX-M are the predominant types that confer resistance to beta-lactam group of antibiotics. Many reports have been investigated ESBL-producing isolates of Enterobacteriaceae in Iraq. However, there are few studies concerned about ESβL-producing Serratia marcescens particularly, detection of ESBLs encoding genes. Therefore this study aimed to identify ESβLs encoding genes in Serratia marcescens isolates from a neonatal intensive care unit. Fifty isolates were identified phenotypically using the VITEK® 2 compact system. For confirming the identification of bacterial strains, molecular detection of housekeeping LuxS gene was done using species-specific designed primers. Antibiogram was performed using the VITEK® 2 compact system. A phenotypic confirmatory test for ESβLs producers was performed using a combination disc method. The ESβLs encoding genes, including blaTEM, blaSHV, and blaCTX-M, were amplified using a PCR-based technique; the amplified products of some selected isolates were sequenced. Molecular detection of isolates using PCR-based amplification of the LuxS gene showed that all isolates possessed this gene. The patterns of antimicrobial resistance for isolates under study showed very high resistance to cephalosporins, while they were susceptible to carbapenem drugs and tigecycline. Findings based on the PCR technique showed that the prevalence of ESβLs encoding genes of isolates was 13 (26%), 31 (62%), and 46 (92%) for blaTEM, blaSHV, and blaCTX-M respectively. In the present study, it was concluded that blaCTX-M gene was the most prevalent ESβLs-encoding gene among ESβLs producing Serratia marcescens isolates.

Validated prognostic significance of YB-1 genetic variation in metastatic prostate cancer.

Genetic polymorphism in YB-1 was previously shown to be associated with the prognosis of advanced prostate cancer patients treated with primary androgen-deprivation therapy. However, the significance of this polymorphism remains invalidated. In this study, we aimed to validate the prognostic significance of the YB-1 genetic polymorphism in metastatic prostate cancer. This study included 79 Japanese patients who were diagnosed as metastatic prostate cancer between 2000 and 2016. Genomic DNA was obtained from patient whole blood samples, and genotyping on YB-1 (rs12030724) was performed by PCR-based technique. The association of genotype in YB-1 with clinicopathological parameters and oncological outcome, including progression-free survival and overall survival, was examined. Homozygous wild-type (AA), heterozygous variant (AT), and homozygous variant (TT) were identified in 47 (59.5%), 26 (32.9%) and 6 patients (7.6%), respectively. Heterozygous/homozygous variant (AT/TT) in YB-1 was significantly associated with lower progression risk compared with homozygous wild-type (AA) (hazard ratio = 0.52; 95% confidence interval = 0.30-0.88, P = 0.015). Consistent with this finding, heterozygous/homozygous variant (AT/TT) in YB-1 was significantly associated with lower risk of any-cause mortality compared with homozygous wild-type (AA) (hazard ratio = 0.46; 95% confidence interval = 0.21-0.93, P = 0.031). Gene polymorphism in YB-1 rs12030724 was validated to be a promising predictive biomarker of androgen-deprivation therapy in metastatic prostate cancer to identify patients requiring more intensive therapeutics.

Taxonomic dysbiosis of gut microbiota and serum biomarkers reflect severity of central nervous system injury

The term “chronic critical illness” (CCI) refers to patients with prolonged dependence on intensive care. In most patients, CCI is triggered by severe brain injury. Ever more studies researching the microbiota in pathologic conditions are published every year, but a lot is yet to be elucidated about the composition of the gut microbiota in CCI. The aim of this study was to investigate possible correlations between changes in the taxonomic abundance of the gut microbiota, levels of proinflammatory and neurological serum biomarkers and the severity of central nervous system injury in patients with CCI. Our prospective observational pilot study included 29 patients with CCI. Using real-time PCR allowed us to detected changes in the taxonomic abundance of the gut microbiota. The correlation analysis of serum biomarkers and the taxonomic composition of the gut microbiota revealed statistically significant correlations between cortisol levels and the abundance of F. prausnitzii (r = ‒0.62; p < 0.05) and B. thetaiotaomicron (r = ‒0.57; p < 0.05) in vegetative state patients; between the CRP/albumin ratio and the abundance of S. aureus (r = 0.72; p < 0.05); between the abundance of B. fragilis group/F. prausnitzii and S100 levels (r = 0.45; p <0.05) in conscious patients; between Glasgow coma scale scores and the abundance of Enterococcus spp. (r = ‒0.77; p <0.05) in both groups. Thus, the association between the changes in the taxonomic composition of the gut microbiota and the severity of neurologic deficit can be evaluated using PCR-based diagnostic techniques and blood serum biomarkers. This approach will help to optimize antibacterial treatment regimens and/or develop alternative strategies to minimize the aggressive effect of antibiotics on the gut microbiota.

A single-cell polony method reveals low levels of infected Prochlorococcus in oligotrophic waters despite high cyanophage abundances

Long-term stability of picocyanobacteria in the open oceans is maintained by a balance between synchronous division and death on daily timescales. Viruses are considered a major source of microbial mortality, however, current methods to measure infection have significant methodological limitations. Here we describe a method that pairs flow-cytometric sorting with a PCR-based polony technique to simultaneously screen thousands of taxonomically resolved individual cells for intracellular virus DNA, enabling sensitive, high-throughput, and direct quantification of infection by different virus lineages. Under controlled conditions with picocyanobacteria-cyanophage models, the method detected infection throughout the lytic cycle and discriminated between varying infection levels. In North Pacific subtropical surface waters, the method revealed that only a small percentage of Prochlorococcus (0.35–1.6%) were infected, predominantly by T4-like cyanophages, and that infection oscillated 2-fold in phase with the diel cycle. This corresponds to 0.35–4.8% of Prochlorococcus mortality daily. Cyanophages were 2–4-fold more abundant than Prochlorococcus, indicating that most encounters did not result in infection and suggesting infection is mitigated via host resistance, reduced phage infectivity and inefficient adsorption. This method will enable quantification of infection for key microbial taxa across oceanic regimes and will help determine the extent that viruses shape microbial communities and ecosystem level processes.

Genetic Distances of Rainbow Trout and Masu Salmon as Determined by PCR-Based Analysis

This study used a PCR-based genetic analysis platform to create a hierarchical polar dendrogram of Euclidean genetic distances for two salmonid species, Oncorhynchus mykiss (rainbow trout, RT) and Oncorhynchus masou (masu salmon, MS). The species were distantly related to other fish species based on PCR results from using the designed oligonucleotide primer series. Five oligonucleotide primers were used to generate 330 and 234 scorable fragments in the RT and MS populations, respectively. The DNA fragments ranged in size from approximately 50 bp to more than 2,000 bp. The bandsharing (BS) results showed that the RT population had a higher average BS value (0.852) than that for the MS population (0.704). The genetic distance between individuals supported the presence of adjacent affiliation in cluster I (RT 01-RT 11). The observation of a significant genetic distance between the two Oncorhynchus species verifies that this PCR-based technique can be a useful approach for individual- and population-based biological DNA investigations. The results of this type of investigation can be useful for species safekeeping and the maintenance of salmonid populations in the mountain streams of Korea.

Targeted Methylation Analyses: From Bisulfite Treatment to Quantification.

DNA methylation constitutes the most studied epigenetic mechanism, regulating gene expression in several physiological and pathological states. Targeted methylation polymerase chain reaction (PCR)-based analyses are among the most universal and commonly used techniques in research. They can be of use for validating methylation-based biomarkers to include in clinical practice. Optimal execution and interpretation of data is fundamental for achieving accurate and reproducible results.In this chapter we describe the backbone procedures behind targeted methylation analyses: bisulfite conversion and downstream PCR-based techniques, including real-time quantitative methylation-specific PCR (qMSP) and high-resolution melting (HRM) methylation-sensitive analyses. Specifically, we give details about the protocol, discuss the pros and cons of these methodologies, and give practical tips for achieving optimal results and for troubleshooting.

The molecular effect of a polymorphic microRNA binding site of Wolfram syndrome 1 gene in dogs

BackgroundAlthough the molecular function of wolframin remains unclear, the lack of this protein is known to cause stress in the endoplasmic reticulum. Some variants in the Wolfram Syndrome 1 gene (WFS1) were associated with various neuropsychiatric disorders in humans, such as aggressiveness, impulsivity and anxiety.ResultsHere we present an in silico study predicting a single nucleotide polymorphism (rs852850348) in the canine WFS1 gene which was verified by direct sequencing and was genotyped by a PCR-based technique. We found that the rs852850348 polymorphism is located in a putative microRNA (cfa-miR-8834a and cfa-miR-1838) binding site. Therefore, the molecular effect of allelic variants was studied in a luciferase reporter system that allowed assessing gene expression. We demonstrated that the variant reduced the activity of the reporter protein expression in an allele-specific manner. Additionally, we performed a behavioral experiment and investigated the association with this locus to different performance in this test. Association was found between food possessivity and the studied WFS1 gene polymorphism in the Border collie breed.ConclusionsBased on our findings, the rs852850348 locus might contribute to the genetic risk of possessivity behavior of dogs in at least one breed and might influence the regulation of wolframin expression.

Successful Leptospira genotyping strategy on DNA extracted from canine biological samples

Leptospirosis is an emerging worldwide zoonosis with a changing epidemiology responsible for an acute disease in humans and dogs. A better knowledge of the responsible bacterium Leptospira and in particular its various serovars and serogroups prevalence is essential for better diagnosis and prevention of the disease. The gold standard for leptospirosis diagnosis is the Microscopic Agglutination Test (MAT) but it requires long and fastidious laboratory work and sometimes results in controversial data. For these reasons, PCR-based techniques for detection of pathogenic leptospiral DNA in biological samples are currently replacing the MAT. However, these strategies do not provide any information regarding the infecting serovar or serogroup. In this study, an optimized genotyping method is described to allow the identification of Leptospira ssp. directly at serovars level using DNA extracted from canine blood and urine. 16S rDNA, Variable Number Tandem Repeat (VNTR) and Multispacer Sequence Typing (MST) protocols were adapted to biological samples. Eighty-eight DNA samples were analyzed from 72 different European canine clinical cases of leptospirosis confirmed by real-time PCR. 92% of DNA samples with Ct values below 34 were fully typed, and typing success decreased to about 30% for the other samples. Typing failure also showed a specie-specific correlation, with 63% of complete typing for L. interrogans and only 40% for L. kirschneri. Additionally, an exact match was observed between serological and molecular data for the few investigated cases where MAT data were available. This methodology is a suitable alternative to the MAT for determining the infecting serovar when Leptospira DNA from blood or urine is detected at Ct values below 34, contributing to clinical surveillance of leptospirosis.

Survey of Wolbachia frequency in Nashville, Tennessee Reveals Novel Infections

Wolbachia (Rickettsiales: Anaplasmataceae) are maternally transmitted intracellular bacteria that infect approximately half of all insect species. These bacteria commonly act as reproductive parasites or mutualists to enhance their transmission from mother to offspring, resulting in high prevalence among some species. Despite decades of research on Wolbachia’s global frequency, there are many arthropod families and geographic regions that have not been tested for Wolbachia. Here, arthropods were collected on the Vanderbilt University campus in Nashville, Tennessee, where Wolbachia frequency has not been previously studied. The dataset consists of 220 samples spanning 34 unique arthropod families collected on the Vanderbilt University campus. The majority of our samples were from the families Blattidae (Blattodea), Pulicidae (Siphonaptera), Dryinidae (Hymenoptera), Aphididae (Hemiptera), Paronellidae (Entomobryomorpha), Formicidae (Hymenoptera), Pseudococcidae (Hemiptera), Sphaeroceridae (Diptera), and Coccinellidae (Coleoptera). PCR-based techniques were used to assign infection states and, from these data, the first cases of Wolbachia in the Paronellidae springtails, Lithobiidae (Lithobiomorpha) centipedes, Lonchopteridae (Diptera) spear-winged flies, Sepsidae (Diptera) black scavenger flies, Cryptocercidae (Blattodea) wood roaches, and Lauxaniidae (Diptera) acalyptrate flies were identified. Within-family infection frequencies ranged from 17-100% when Wolbachia was observed; however, numerous families tested did not reveal evidence of infection. These results expand on the field’s understanding of Wolbachia’sfrequencyin Nashville, Tennessee, and among arthropod families broadly, and is the first report of Wolbachia in centipedes. KEYWORDS: Wolbachia; Infection Frequency; Endosymbiont; Tennessee; Centipede; Arthropod; Polymerase Chain Reaction; Nashville

Fast and Accurate Pneumocystis Pneumonia Diagnosis in Human Samples Using a Label-Free Plasmonic Biosensor.

Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivity and reproducibility, achieving limits of detection of around 2.11 nM. A preliminary evaluation of clinical samples showed rapid, label-free and specific identification of P. jirovecii in human lung fluids such as bronchoalveolar lavages or nasopharyngeal aspirates. These results offer a door for the future deployment of a sensitive diagnostic tool for fast, direct and selective detection of Pneumocystis pneumonia disease.

Microscopic and PCR-based detection of microsporidia spores in human stool samples

Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrhea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Genetic variants association with cancers in African-based populations: A systematic review

BackgroundCancer is the single leading cause of human deaths worldwide. The highest incidence and mortality are recorded from Africa. The last two decades have witnessed extensive research which has led to emerging prognosis and new gene therapy technologies. Cancer therapy in Africa is derived with little input from African population data. While a number of cancer studies on African populations have suggested varied susceptible variant, no comprehensive review of these studies has been undertaken to assess their coverage across Africa. MethodsThis study aimed to undertake a review of all molecular genetic studies that interrogated the genetic variants of cancers in African-based populations. Our search methodology was modelled after the Cochrane systematic review protocol, which included MeSH terms and related keywords. ResultsNinety-seven articles studying 13 cancer types, were reviewed. 91 articles screened for polymorphisms using PCR-based techniques while three used SNP array, two used whole exome sequencing and one used pyrosequencing. North African (NA) countries undertook 51/97 (53 %) studies on 12/13 (92 %) cancer types while the Sub Saharan Africa (SSA) countries undertook 46/97 (47 %) studies on 7/13 (54 %) cancer types. Twelve out of these thirteen cancer type studies suggested susceptibility to their target polymorphism (p > 0.05). No study replicated or validated variants detected. ConclusionResearch on genetic determinants in African-based population cancer offers translational benefits. We recommended large scale, multi-national genome association studies using high throughput techniques. SSA needs to receive more attention due to the shortage of this type of study and data in the region.

Abstract A13: DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA

Abstract Background: Accumulated evidence revealed that aberrant CpG island hypermethylation plays significant roles in carcinogenesis that can serve as a promising target for molecular detection in body fluids. Ovarian cancer remains the most lethal gynecologic malignancy and is characterized by few early symptoms, late stage presentation, and resulting poor survival, which thereby renders challenge to its prompt diagnosis. Despite a myriad of attempts at early diagnosis of ovarian cancer, this clinical aim still remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant circulating DNA methylation patterns provide highly specific cancer signals. Methods: To develop a DNA methylation-based screening assay for early diagnosis of ovarian cancer, we quantitatively assessed the promoter methylation of DAPK1, HOXA9, RASSF1A, and SOX1 genes in 105 ovarian cancer and 30 non-neoplastic ovarian specimens by means of a high-throughput quantitative, real-time PCR-based technique (MethyLight) and clonal bisulfite sequencing. The best-performing gene cassette was further evaluated for their methylation status in cell-free DNA from serum of matched 35 ovarian cancer patients and 20 normal controls. Area under the ROC (Receiver-operator characteristic) curve was used to evaluate the discriminatory performance of both individual and combined gene panel. Results: RASSF1A, HOXA9, DAPK1, and SOX1 displayed a methylation frequency of 72.72, 82.67, 68.0, and 80.0%, respectively, in tissue samples. We identified DNA methylation of HOXA9 and SOX1 as the best discriminator between cancer and non-neoplastic tissue. In the multiplex assay, the combined panel of HOXA9 and SOX1 achieved a sensitivity of 88.0% with a specificity of 86.7%, when either or both of the gene promoters showed methylation. These genes appear to have great potential to be evaluated for their methylation level in cell-free DNA as a noninvasive diagnostic marker for early diagnosis of ovarian cancer. In cell-free DNA, this novel panel achieved a sensitivity of 62.86.0% and a specificity of 95% with an area under the ROC curve of 0.81 for discriminating OC samples from normal control. The results of MethyLight were concordant with those of clonal bisulfite sequencing. Conclusion: Our findings revealed the potential of epigenetic biomarker for early detection of OC in cell-free DNA as a minimally invasive tool. Here, we have established that promoter hypermethylation can be detected in serum cell-free DNA from patients with early as well as advanced-stage ovarian cancer. Based on the multiplex MethyLight assay, study will focus on analyzing the clinical utility of HOXA9 and SOX1 as methylation biomarkers. These findings further underline the potential of multiplexing individual markers into a panel that helps to achieve higher sensitivity and specificity than individual marker and provides robustness to a noninvasive early screening test. Citation Format: Alka Singh, Jaydeep Aravindbhai Badarukhiya, Sameer Gupta, Manisha Sachan. DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A13.

Antimicrobials and Enterobacterial Repetitive Intergenic Consensus (ERIC) Polymerase Chain Reaction (PCR) Patterns of Nosocomial Serratia Marcescens Isolates: A One Year Prospective Study (June 2013-May 2014) in a Rural Hospital in the Republic of Trinidad and Tobago

Background: Serratia marcescens is a gram-negative bacterium from family of Enterobacteriaceae. It is a human pathogen that is involved in nosocomial infection outbreaks that have proven difficult to manage. PCR-based techniques are suitable given the genus. Serratia has a higher GC content than many other members of the Enterobacteriaceae. The ERIC PCR-based fingerprinting method was used to study the PCR patterns of clinical Serratia marcescens isolates and antimicrobial susceptibility profiles. Methods: Surveillance was conducted for nosocomial cases of interest and the nosocomial pathogens were retrieved for identification via morphological and additional biochemical tests. Additionally, antimicrobial susceptibility tests and ERIC Polymerase Chain Reaction (PCR) based fingerprinting molecular method was done on the clinical S. marcescens isolates. Results: From the study, five different strains of the clinical Serratia marcescens isolates were recovered and similarly five distinct susceptibility patterns were observed from the clinical S. marcescens isolates indicating consistency in the number of strains present in the clinical S. marcescens isolates. Conclusions: ERIC PCR fingerprinting base technique; a simple, rapid and cheap method for the determination of genetic relatedness between Serratia marcescens isolates can be applied for the thorough evaluation of nosocomial outbreaks to detect the source of infection and control the spreading of the infection.