Polymerase Chain Reaction Reaction Research Articles

Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver

Background & AimsHepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear.MethodsWe performed studies with BALB/c Rag2–/–Il2rg–/–SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells.ResultsFollowing HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation.ConclusionIn HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

ObjectiveLower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. MethodsThree multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. ResultsThe detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10−7 to 10−2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. ConclusionThis study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.

Detection and identification of azithromycin resistance mutations on <em>Treponema pallidum</em> 23S rRNA gene by nested multiplex polymerase chain reaction

Background: Azithromycin-resistant strains of Treponema pallidum is associated with the mutation of 23S rRNA gene of T. pallidum. Although these strains are now prevalent in many countries, there is no laboratory test kit to detect and identify these mutations. Thus, in this study we developed a nested multiplex polymerase chain reaction (PCR) to detect and identify A2058G and A2059G mutations in 23S rRNA gene.Methods: Three primer sets were designed for nested PCR reactions. To obtain maximum PCR reaction, all parameters were optimized. The specificity of the primer sets was evaluated towards some microorganisms. A sensitivity test was conducted to get detection limit of deoxyribonucleic acid (DNA). Forty-five whole blood specimens were tested by PCR, and positive results were confirmed by the DNA sequencing.Results: The assay could detect at least 4,400 DNA copy number and showed no cross reaction with other microorganisms used in the specificity test. A total 13 of 45 whole blood specimens were PCR positive for T. pallidum, and no single mutations (either A2058G or A2059G) were detected. Two positive specimens were confirmed by the DNA sequencing and showed no mutation.Conclusion: Nested multiplex PCR developed in this study showed a specific and sensitive test for the detection and identification of A2058G and/or A2059G mutations of 23S rRNA T. pallidum gene.

Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR

ObjectiveAccurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications. Materials and methodsTo formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification. ResultsAnalysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 106-fold dilution of primary PCR products. ConclusionThe SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic.

Comparative study of DNA extraction methods from fresh and processed yellowfin tuna muscle tissue

ABSTRACTThe most common techniques used to identify tuna species include methods based on the detection of specific DNA. The quality of species-specific DNA crucially affects the efficiency of amplification during the subsequent polymerase chain reaction (PCR). Detection of DNA in processed products can be adversely affected by DNA fragmentation during the processing steps and the use of ingredients that may inhibit the PCR reaction. In this study, several processing treatments applied to the muscle tissue of yellowfin tuna (Thunnus albacares) were evaluated. For DNA isolation three DNA extraction methods were compared. The concentration, purity, and amplificability of DNA were tested. The results revealed the variability among extraction procedures in terms of DNA quality and quantity in tuna muscle tissue processed under different processing technologies.

Heart failure and endothelial nitric oxide synthase G894T gene polymorphism frequency variations within ancestries

The G894T polymorphism in endothelial nitric oxide synthase enzyme gene plays an important role in heart failure (HF) and its frequency varies among populations. We investigated this association in highly admixed samples in terms of ancestry. The cohort included 210 HF patients and 106 healthy individuals. Self-reported race and NYHA class were analyzed for HF patients. G894T polymorphism was analyzed by polymerase chain reaction (PCR) and by restriction fragment length polymorphism technique. Ancestry was estimated using a PCR reaction containing 46 autosomal ancestry informative markers and an analysis by capillary electrophoresis. The GG homozygous genotype had a higher frequency in HF patients (63.8%) than in healthy individuals (48.1%), showing an increased chance (odds ratio 1.90, 95% confidence interval 1.18–3.05). The ancestry profiles in patients and controls were similar, with a major European contribution (57.1% and 63.2%), followed by African (30.2% and 24.0%) and Native American (12.7% and 12.8%), without a significant difference between both samples (p = 0.28). The GG genotype is associated to HF prognosis, and this association remains present in highly admixed sample groups.

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

AbstractStripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.

Spectrum of Diarrhoeagenic Escherichia coli in Paediatric Population Suffering from Diarrhoea and as Commensals in Healthy Children

Background: Diarrhoeagenic Escherichia coli (DEC) is associated with early death of children in developing countries and are being identified now as an important evolving pathogen. The objective of this study was to perform multiplex polymerase chain reaction (PCR) for simultaneous detection of six categories of DEC in two sets of PCR reactions using 11 virulent genes. Materialsand Methods: During 1-year study period, forty isolates each from outpatient, inpatient and healthy groups were collected from children. E. coli was identified using conventional biochemical methods. DNA extraction was done using kit, and the extracted DNA was used as a template for multiplex PCR. Results: Virulent genes of DEC were detected in 106 (88.33%) samples. Overall, elt and est were detected in 8.33% and 30.83% of specimens; typical, atypical enteropathogenic E. coli and bfp were detected in 13.33%, 29.16% and 19.16% specimens; eagg was detected in 39.16% and east in 13.33% specimens and stx and hyla were isolated in 1.66% specimens each. While diffusely adherent E. coli and enteroinvasive E. coli genes were not isolated. Conclusion: Multiplex PCR is a rapid method for the simultaneous detection of 11 virulent genes of DEC at a time and it will provide a platform in understanding the diarrheal diseases in a more improved manner.

The clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato-oncology unit.

Although many reports have already shown RSV outbreaks among hemato-oncology patients, genomic studies detecting similar RSV strains prior to an outbreak in the hospital are rare. In 2014, the University of the Ryukyus hospital hemato-oncology unit experienced, and successfully managed, a respiratory syncytial virus (RSV) nosocomial outbreak. During the outbreak investigation, genotyping and phylogenetic analysis was used to identify a potential source for the outbreak. Nasopharyngeal swabs were tested for RSV using three tests: (1) rapid antigen test (RAT); (2) reverse transcriptase polymerase chain reaction (PCR); or (3) quantitative PCR (RT-qPCR); a positive PCR reaction was considered a confirmed case of RSV. Phylogenetic analysis of the G protein was performed for outbreak and reference samples from non-outbreak periods of the same year. In total, 12 confirmed cases were identified, including 8 hemato-oncology patients. Patient samples were collected weekly, until all confirmed RSV cases returned RSV negative test results. Median time of suspected viral shedding was 16 days (n = 5, range: 8-37 days). Sensitivity and specificity of the RAT compared with RT-qPCR were 30% and 91% (n = 42). Phylogenetic analysis revealed nine genetically identical strains; eight occurring during the outbreak time period and one strain was detected 1 month prior. A genetically similar RSV detected 1 month before is considered one potential source of this outbreak. As such, healthcare providers should always enforce standard precautions, especially in the hemato-oncology unit.

Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

Polymerase chain reaction (PCR) has been one of the principal techniques of molecular biology and diagnosis for decades. Conventional PCR platforms, which work by rapidly heating and cooling the whole vessel, need complicated hardware designs, and cause energy waste and high cost. On the other hand, partial heating on the various locations of vessels to induce convective solution flows by buoyancy have been used for DNA amplification in recent years. In this research, we develop a new convective PCR platform, capillary loop convective polymerase chain reaction (clcPCR), which can generate one direction flow and make the PCR reaction more stable. The U-shaped loop capillaries with 1.6 mm inner diameter are designed as PCR reagent containers. The clcPCR platform utilizes one isothermal heater for heating the bottom of the loop capillary and a CCD device for detecting real-time amplifying fluorescence signals. The stable flow was generated in the U-shaped container and the amplification process could be finished in 25 min. Our experiments with different initial concentrations of DNA templates demonstrate that clcPCR can be applied for precise quantification. Multiple sample testing and real-time quantification will be achieved in future studies.

Small Ruminant Lentiviruses in Sheep: Pathology and Tropism of 2 Strains Using the Bone Marrow Route.

The objective of this work was to comparatively study the tissue tropism and the associated pathology of 2 autochthonous small ruminant lentivirus (SRLV) field strains using an experimental infection in sheep through the bone marrow. Fifteen male, SRLV-free lambs of the Rasa Aragonesa breed were inoculated with strain 697 (nervous tissue origin, animals A1-A6), with strain 496 (articular origin, animals B1-B6), or with uninfected culture medium (C1-C3). Clinical, serologic, and polymerase chain reaction (PCR) evaluations were performed periodically. Two lambs from each infected group and a control animal were euthanized at 134, 273, and 319 days postinfection. Tissues were analyzed by gross and histopathologic evaluation; immunohistochemistry for CD3, CD4, CD8, CD68, and FoxP3 cell markers; lung morphometric evaluation; and tissue proviral quantification by PCR. All infected animals became positive either by enzyme-linked immunosorbent assay and/or PCR, with group B lambs showing the highest serologic values and more consistently positive PCR reactions. Group A lambs showed representative lung lesions but only mild histopathologic changes in the central nervous system (CNS) or in carpal joints. Contrarily, group B lambs demonstrated intense carpal arthritis and interstitial pneumonia but an absence of lesions in the CNS. Proviral copies in tissues were detected only in group B lambs. Experimental infection with these SRLV strains indicates that strain 496 is more virulent than strain 697 and more prone to induce arthritis, whereas strain 697 is more likely to reproduce encephalitis in Rasa Aragonesa lambs. Host factors as well as viral factors are responsible for the final clinicopathologic picture during SRLV infections.

Real time plasmonic qPCR: how fast is ultra-fast? 30 cycles in 54 seconds.

Polymerase Chain Reaction (PCR) is a critical tool for biological research investigators but recently it also has been making a significant impact in clinical, veterinary and agricultural applications. Plasmonic PCR, which employs the very efficient heat transfer of optically irradiated metallic nanoparticles, is a simple and powerful methodology to drive PCR reactions. The scalability of next generation plasmonic PCR technology will introduce various forms of PCR applications ranging from small footprint portable point of care diagnostic devices to large footprint central laboratory multiplexing devices. In a significant advance, we have introduced a real time plasmonic PCR and explored the ability of ultra-fast cycling compatible with both label-free and fluorescence-based monitoring of amplicon production. Furthermore, plasmonic PCR has been substantially optimized to now deliver a 30 cycle PCR in 54 seconds, with a detectable product. The advances described here will have an immediate impact on the further development of the use of plasmonic PCR playing a critical role in rapid point of care diagnostics.

Development of a new multiplex-PCR tool for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda

Disease assessment and management in cultured aquatic animals is a major concern in commercial aquaculture. Disease outbreaks have direct effects on fish production, causing serious economic losses in this industry. This can be overcome by early detection through molecular high sensitivity tools such as PCR (polymerase chain reaction). One of the most critical steps in the study of bacterial fish diseases is the precise identification of the infectious agent. Considering the damage that some bacteria can cause to fish and humans, the development of a rapid detection method for the four target species that demonstrates to be simple, accurate and low cost is an essential step for the prevention and early treatment of these diseases. Edwardsiella tarda (ACC 36.1), Vibrio harveyi (DSM 19023), Vibrio anguillarum (AQV 55.1) and Vibrio alginolyticus (CECT 521) were selected as targets of a multiplex PCR tool. The multiplex PCR reactions were performed in various reaction conditions, including different annealing temperatures (between 49 °C and 55 °C) and changes in the MgCl2 concentration from 2 mM to 8 mM. Best results were obtained at 51 °C and MgCl2 concentration from 4 mM to 6 mM. Primers were tested using purified DNA from the respective bacterial strains, yielding bands at expected sizes, which can be identified on agarose gels with clearness and without overlapping sizes. Results indicate that this multiplex PCR tool is suitable for the detection of target pathogens and may, in the future, have extended practical application in commercial aquaculture.

Characterization of Barley Yellow Mosaic Virus Pathotype by Biological and Molecular Methods in Iran = وصف سلالات فيروس تبرقش وإصفرار الشعير باستخدام الطرق البيولوجية والجزيئية في إيران

Barley yellow mosaic virus (BaYMV) is one of the economically important viruses infecting cereals. Different pathotypes of BaYMV divided in four pathotypes (I to IV) according to reaction on cultivars carrying different rym resistance genes. From March to April 2014,141 leaf samples of barley collected from four main growing areas of cereals in Iran. Samples were tested by double-antibody sandwich enzyme-linked immunosorbent assays (ELISA) using polyclonal antisera and also polymerase chain reaction (PCR). In order to determine BaYMV pathotype in Iran we investigated reaction of Iranian pathotypes of BaYMV on different barley cultivars. We also used specific primers in PCR reaction amplifying 690 bp of the VPg coding region, one of the important genes involved in viral pathogenicity. The amplified fragments from 5 isolates selected according to their geographical proprieties,were cloned and then sequenced. Biological properties and VPg sequence comparison of Iranian isolates with others retrieved from databases suggested that Iranian isolates are closely related to pathotype I of BaYMV. Nevertheless, some changes in the amino acid sequences of VPg of Iranian isolates might affect the biological behavior on cultivars carrying other rym resistant genes. According to our knowledge, this research is the first study of BaYMV pathotype in Iran.

Bacterial profiling of healthy bone marrow using polymerase chain reaction technique

Background: The bone marrow of healthy individuals is conventionally considered sterile like other body fluids, but recently the scientists found genetic material from the bacteria inside their stem cells. The findings raise the possibility that other infectious agents may also reside in the bone marrow. Objectives: The study was planned to look for the presence of all type of bacteria by amplifying 16S rDNA sequences using eubacterial universal primers. Materials and Methods: A total of 18 bone marrow samples of apparently healthy individuals were collected from patients admitted having closed bone fractures. The DNA was isolated and was subjected to nested polymerase chain reaction (PCR) using Universal eubacterial 16S rDNA primers. The samples positive by universal PCR was further checked for the presence of Salmonella Typhi, Salmonella Paratyphi A, and/or Mycobacterium tuberculosis (Mtb) if any using a second nested PCR reaction. Results: A total of 16 (89%) samples could yield the desired amplicon through universal PCR. The secondary PCR of 16 samples, the desired amplicons were detected 3 (18.8%) for Mtb, 4 (25%) for S. Typhi, and 1 (6.2%) for S. Paratyphi A. Conclusions: Even in asymptomatic cases, other infectious agents such as Mtb, Salmonella spp., and many other microorganisms may be present as commensal.

RT PCR Data Plagiarism Their Ignorance and Impact

Polymerase Chain Reaction (PCR) is a technique based on the ability of DNA polymerase to synthesize a new strand of DNA which is complementary to the template strand offered and amplifying this strand to billions of amplicons. There are different types of PCR reactions for different types of experiments but reverse transcription-polymerase chain reaction (RT-PCR) is most commonly used PCR reactions in the field of genomics and proteomics basically at mRNA level. A wide range of procedures have been devised for studying PCR products like gel electrophoresis, cloning and sequencing of PCR products, etc. The PCR products from gel electrophoresis are in the form of images, therefore image processing techniques are commonly used to analyze these images in three main steps i.e. band detection, band matching, and data quantification. Image processing sometimes alters the intensity of image which can at points improve the visualization of the data. But if that image alteration mischaracterizes the data, one has gone too far and this is known as plagiarism. Therefore, it is important to safeguard and protect the original, unaltered image in order to avoid the accusations of misconduct which will stand or fall on the basis of whether or not the original image is available to be compared with its altered copies. For example, investigators whose work fall under the food and drug administration’s (FDA’s) “Final Rule on Electronic Records and Electronic Signatures” must maintain the integrity of the original image. Similarly, industries whose work products are used in forensic activities or in health insurance portability and accountability act (HIPAA) related aspects of health care might be required to maintain an original image. Therefore, the plagiarism of RT-PCR data has tremendous impact on research if left ignored as seen through the above examples and therefore should be taken care.

A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

BackgroundProgressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs.ResultsIn comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR.ConclusionsThe approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13028-016-0267-7) contains supplementary material, which is available to authorized users.

Detection of Diarrhea Etiology Among U.S. Military Personnel During Exercise Balikatan 2014, Philippines, Using TaqMan Array Cards.

Military personnel are vulnerable to diarrhea. Diarrheal disease is common when deployed for operations or exercise in developing countries. Although diarrheal disease is transient, cumulative time lost and medical asset can have a significant impact on military operations. Currently, diagnostics of diarrheal etiology typically relies on a mixture of conventional bacteriology, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR)-based methods including real-time PCR. These methods, however, can be time and labor intensive, although the identification of diarrheal etiology needs to be informative and rapid for treatment and prevention. Real-time PCR has been increasingly used to identify pathogens. Real-time PCR panels of common diarrheal pathogens have been developed, but several diarrheal pathogens are not included in the panel. An expanded and customizable panel to detect diarrhea etiology has been developed employing TaqMan Array Card (TAC) technology. TAC performs 384 real-time PCR reactions simultaneously. As currently configured for diarrheal disease by the University of Virginia, a maximum of 8 samples can be tested simultaneously with approximately 48 target pathogens per sample including bacteria, fungi, helminths, protozoan parasites, and viruses. TAC diarrheal disease panels have been successfully applied to detect pathogens in acute diarrheal stool samples from young children in several international multicenter diarrhea studies. In this study, TAC was applied to stool samples collected under an approved human use protocol from military personnel with acute diarrhea participating in the annual joint military exercise, Balikatan, between the Republic of the Philippines and the United States in 2014. Several established pathogen-specific real-time PCR detection assays were also performed in parallel for comparative purposes. TAC was applied to 7 stool samples. Campylobacter spp. was the most common diarrheal disease pathogen detected. Results from TAC matched 5 out of 6 pathogen specific real-time PCR assays. TAC required a total of 5-6 hours to complete all the procedures from nucleic acid extraction and data analysis, whereas a minimum of 18 hours and 4 hours are required for conventional bacteriology and enzyme-linked immunosorbent assay, respectively, per pathogen. With TAC, pathogen load can be estimated from the amount of nucleic acid present for each pathogen, which can be analyzed further to better determine pathogen attribution and to compare pathogen load between case and control samples. Unfortunately, such correlative analysis was not possible because of the limited sample size available in this study. A larger sample size is needed for further evaluation of TAC on a specific population set, including military personnel. Regardless, TAC was found to be a useful and functional diagnostic platform that is less time-consuming, easy to use with high reproducibility, and costs less per sample compared to the current typically employed methods. The successful application of TAC in acute diarrhea stool samples from a US military population in the Philippines demonstrates its versatility as a potential candidate for a next-generation diagnostics platform.

Comparison of Five Commercial Nucleic Acid Extraction Kits for the PCR-based Detection of Burkholderia Pseudomallei DNA in Formalin-Fixed, Paraffin-Embedded Tissues.

The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR.FFPE samples from organs of Burkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targeting B. pseudomallei-specific sequences of varying length after 16 years of storage.The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal.The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits.

Abstract 3186: Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR

Abstract Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.