Polymerase Chain Reaction Technology Research Articles

Hippocampal neuron loss and astrogliosis in medial temporal lobe epileptic patients with mental disorders.

Hippocampal neuron loss and reactive astrogliosis are pathological features of medial temporal lobe epilepsy. Here, the expression of hippocampal astrogliosis-associated genes are studied in subjects with medial temporal lobe epilepsy and mental disorders (such as depression, anxiety and psychiatric comorbidities). The relationship between functional changes in hippocampus astrocytes and concurrent mental disorders are discussed. Nissl staining identified medial temporal lobe epilepsy-induced neuronal loss in the CA1 region of hippocampus. Quantitative real-time polymerase chain reaction and immunofluorescence technology were used to detect hippocampus glial fibrillary acidic protein, metallothionein, and aquaporin-4. The hippocampus area of subjects with medial temporal lobe epilepsy (with or without mental disorders) were smaller than the control group. Hippocampal neuronal loss and astrogliosis were more obvious in groups of medial temporal lobe epileptic patients with mental disorders. Relative protein levels of glial fibrillary acidic protein, metallothionein-I/II, and aquaporin-4 were significantly higher in subjects with medial temporal lobe epilepsy than seen in controls. Medial temporal lobe epileptic patients with mental disorder or depression had elevated metallothionein-I/II protein level when compared to controls and medial temporal lobe epileptic patients without mental disorder. Protein levels of glial fibrillary acidic protein and aquaporin-4 in medial temporal lobe epileptic patients with mental disorders were significantly lower than that in medial temporal lobe epileptic patients with no mental disorder. It is concluded that functional changes in hippocampus astrocytes are associated with mental disorders in medial temporal lobe epileptic patients and the astrogliosis-related genes of glial fibrillary acidic protein, metallothionein-I/II and aquaporin-4, are involved in this process.

The relationship between human papillomavirus and penile cancer over the past decade: a systematic review and meta-analysis.

Human papillomavirus (HPV) infection appears to play an important role in the development of penile cancer (PeCa), but their relationship remains unclear. Therefore, we performed a systematic review and meta-analysis to elucidate their relationship. We systematically searched Embase, PubMed, Cochrane Library, and Web of Science for case-control studies and cross-sectional studies using polymerase chain reaction (PCR) technology on formalin-fixed paraffin-embedded (FFPE) or paraffin-embedded (PE) PeCa tissues to detect HPV (published between January 1, 2007, and December 29, 2017; no language restrictions). Twenty-two studies were identified, and 1664 cases were available for analysis. The combined HPV infectious risk of PeCa is 51.0% (95% confidence interval [CI]: 43.0%–60.0%). The three most common subtypes of HPV were HPV16 (28.5%), HPV18 (2.3%), and HPV6 (2.3%). The virus was relevantly associated with basaloid (85.5%, 95% CI: 77.2%–93.8%) and warty (50.0%, 95% CI: 35.2%–64.8%) carcinomas. The invasiveness of PeCa was not associated with HPV (χ2 = 0.181, df = 1, P < 0.671). HPV infection in PeCa tended to be moderately differentiated (54.4%, 95% CI: 47.7%–61.1%). This study found that almost half of PeCa patients are associated with HPV. The most commonly associated genotype is HPV16, but several other genotypes were also detected. In addition to types 6 and 11, other single low-risk HPV infections have been found to contribute to PeCa to a lesser degree. HPV-positive tumors tend to exhibit warty and/or basaloid features, corresponding to a moderate histological grade. The role of HPV in PeCa should be revisited to provide evidence for the development of PeCa in the presence of HPV infection.

TWEAK/Fn14 Is Overexpressed in Crohn's Disease and Mediates Experimental Ileitis by Regulating Critical Innate and Adaptive Immune Pathways.

Background & AimsCrohn’s disease (CD) is a debilitating inflammatory disorder that affects more than 1.6 million people in North America alone. Members of the tumor necrosis factor superfamily are key regulators of intestinal inflammation; specifically, tumor necrosis factor–like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor–inducible 14 (Fn14), are involved in normal and pathologic tissue remodeling. Our aim was to determine the role of TWEAK/Fn14 in CD and a murine model of CD-like ileitis (ie, SAMP1/YitFc [SAMP] strain).MethodsSAMP mice deficient in Fn14 (SAMP × Fn14-/-) were developed and a detailed time-course study was performed evaluating ileal tissues by histology and stereomicroscopy, as well as quantitative polymerase chain reaction and NanoString technology (Seattle, WA). Reciprocal bone marrow chimeras were generated to assess the relevance of Fn14 in hematopoietic vs nonhematopoietic compartments. Surgically resected intestinal tissues and mucosal biopsy specimens from patients with CD, ulcerative colitis, and healthy controls were analyzed for the expression of TWEAK/Fn14 by quantitative polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence.ResultsSAMP × Fn14-/- showed a marked decrease in ileitis severity at 20 weeks of age compared with SAMP WT controls. Bone marrow chimeras showed that Fn14 was required in both hematopoietic and nonhematopoietic compartments for ileitis to develop. Transcriptome data showed multiple cellular pathways regulated by Fn14 signaling. Finally, increased expression of TWEAK and Fn14 was observed in tissue lesions from CD patients compared with ulcerative colitis and healthy controls.ConclusionsTWEAK/Fn14 are up-regulated in CD, and also mediate experimental CD-like ileitis, by regulation of multiple innate and adaptive cellular pathways. Therefore, TWEAK/Fn14 may represent a novel therapeutic target for the treatment of small intestinal inflammation in CD.

Rapid identification of medicinal leech by species-specific polymerase chain reaction technology

Background: Leech is a well-known animal-derived Chinese medicine; intentionally or unintentionally, interfusing fake products have exposed patients to high risk. Objective: In this study, we would like to find a rapid and accurate technology to identify medicinal leech. Materials and Methods: Three sets of species-specific primer were designed, and polymerase chain reaction (PCR) conditions were optimized for the four species analyzed. Specificity and sensitivity test were carried out, and the reference sample mixtures were analyzed. Finally, the developed method was used to assess the authenticity of commercially available products. Results: Three sets of species-specific primer were confirmed to have high interspecies specificity and good intraspecies stability. The limit of detection was estimated to be 1 ng for all of four assayed species. Subsequent validation demonstrated that the raw material, processed, and even highly processed products of leech can be conveniently authenticated with good sensitivity and precision by this newly established PCR-based method. Conclusion: These designed novel primer sets have shown distinct performance on the identification of adulterate products. The new technology needs no procedure for sequencing and has been successfully employed for the reliable authentication of raw material of leech and even their processed and highly processed products.

Detection and quantification of black foot and crown and root rot pathogens in grapevine nursery soils in the Western Cape of South Africa

Black foot disease (BFD) and crown and root rot (CRR) are important soilborne diseases that affect young grapevines in nurseries and vineyards. A 3-year survey (2013–2015) of five open-field grapevine nurseries was conducted in the Western Cape Province of South Africa. The survey involved the isolation of BFD and CRR pathogens from grafted rootstocks (ten plants per nursery, per year) that were rooted in soil for 1 year. In 2013 and 2015, grapevines were sampled, while in 2014, sampling was focused on rotation crops and weeds (ten plants each). The rotation crops included white mustard, lupins, canola, triticale and forage radish. The weed species sampled included Johnson grass, ryegrass, winter grass, Cape marigold and corn spurry. Soil samples from ten sites per nursery were also collected in close proximity to the sampled plants, at depths of 0–30 cm and 30–60 cm (ten samples per depth). Isolations were made from the grapevines, rotation crops and weeds. Pathogen detection and quantification in the soil were determined using quantitative real-time polymerase chain reaction technology. The predominant BFD pathogens isolated from grapevines were Campylocarpon fasciculare , Ca. pseudofasciculare and Dactylonectria macrodidyma . The predominant CRR pathogens were Pythium irregulare and Phytopythium vexans. Dactylonectria macrodidyma , D. novozelandica , D. pauciseptata , Py. irregulare , Py. ultimum var. ultimum and Py. heterothallicum were isolated from triticale roots. Dactylonectria spp. were also isolated from corn spurry, while Py. irregulare and Py. ultimum var. ultimum were isolated from numerous weeds and rotation crops. Mean soil DNA concentrations of Ilyonectria and Dactylonectria were from 0.04 to 37.14 pg μL -1 , and for Py. irregulare were between 0.01 and 3.77 pg μL -1 . The Phytophthora mean soil DNA concentrations ranged from 0.01 to 33.48 pg μL -1 . The qPCR protocols successfully detected and quantified BFD and CRR pathogens in grapevine nursery soil. This is the first report of D. pauciseptata and D. alcacerensis in South African grapevine nurseries.

USING QF-PCR ASSAY IN DETECTION OF COMMON GENETIC CAUSES IN INFERTILE MALES

At present, quantitative fluorescent - polymerase chain reaction (QF-PCR) technology is widely being used in prenatal diagnosis of some common genetic syndromes such as Down syndrome, Patau syndrome and Edwards syndrome. The advantages of this technique are its high accuracy, rapid test results and wide applicability. It has used this technique to detect AZF (Azoospermia factor) micro-deletions by Devyser's kit at Tu Du Hospital, Ho Chi Minh City. The novelty of the study was to create a kit with 14 genetic markers and to develop a QF-PCR protocol to detect some common genetic causes in men seeking medical for their infertility with the sperm concentration of ≤ 5 million/ml. We conducted an evaluation in reliability in order to confirm whether the QF-PCR results from the set-up protocol were accurate and reliable. We developed the research on the application of QF-PCR to test for the DNA samples of patients. They were 10 normal reproductive men, 2 patients with Klinefelter syndrome, 4 patients with AZFc micro-deletions, 1 female with normal reproduction. Cordially, we used a sample of water to control the experiment. The results have shown that the kit with 14 genetic markers and the built-in QF-PCR protocol for detecting some common genetic causes in men seeking medical care for their infertility were accurate 100% compared to multiplex-PCR and peripheral blood lymphocyte cultures. The study results are important for identifying some common genetic causes in men seeking medical care for their infertility and helping doctors build the most inferior treatment regimens for their patients.

Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

To assess the effects of food processing on the detection and quantification of transgenic rice TT51-1 in processed food by polymerase chain reaction (PCR) technology, we monitored the presence of TT51-1 components in rice crackers at different processing stages using conventional PCR, quantitative real-time PCR (qPCR), and droplet digital PCR (ddPCR) with standard or validated primers and probes. In conventional PCR, relatively longer amplification targets, such as the Bacillus thuringiensis (Bt) gene (301 bp) and the event-specific target (274 bp), were barely detected in baked, fried or microwaved samples. In qPCR, the amplification fluorescence signal was detected in boiled, dried, baked, and microwaved samples, but barely observed in fried samples. Conventional PCR with the same primers used in qPCR detected the corresponding shorter targets in all samples. The conventional PCR results were mainly consistent with the results of qPCR. The results indicate that food processing directly affects the detection of transgenic components, and suggest that relatively shorter fragments should be selected as the amplification targets for this type of analysis. We established qPCR and duplex ddPCR methods for quantifying TT51-1. The results of an orthogonal experiment indicated that the optimal conditions for TT51-1/PLD duplex ddPCR were 500/250 nM of primers/probe combined with 58 °C annealing temperature. Both methods were feasible for quantitative detection of TT51-1 in processed samples, with duplex ddPCR being a more attractive method for detecting transgenic components in processed food due to its stability, accuracy, PCR inhibitor resistance, and the lack of a need for reference materials.

342. Time to Antimicrobial De-escalation/Discontinuation After Implementation of Cerebrospinal Fluid Polymerase Chain Reaction Tool

BackgroundCerebrospinal fluid (CSF) polymerase chain reaction (PCR) technology can be used as a rapid diagnostic tool that has the potential to more rapidly facilitate targeted antimicrobial therapy and reduce overall time to de-escalation and/or discontinuation of inappropriate antimicrobial usage in patients with suspected meningitis/encephalitis.MethodsThis was a single-center, retrospective cohort analysis with a primary objective focusing on time to de-escalation or discontinuation of inappropriate antimicrobials before and after implementation of a rapid diagnostic meningitis/encephalitis (ME) panel (BioFire FilmArray®). The pre-implementation group, containing 84 patients, examined individuals who had CSF cultures performed in the 6-months prior to implementation. The post-implementation group, containing 88 patients, examined individuals who had an ME panel done in the 6 months following a transitionary 1-month period following implementation. Categorical data analysis was performed using χ2 or Fisher’s exact test and continuous data was analyzed using the Mann–Whitney U test.ResultsTime to de-escalation/discontinuation of inappropriate ampicillin reported in median hours (IQR) was 47.5 (55) for pre-PCR group compared with 39.5 (23.5) in post-PCR group (P = 0.004). Time to de-escalation/discontinuation of Cefotaxime for pre-PCR group was 50.5 (42) compared with 45 (10) for post-PCR (P = 0.027). Using a subgroup analysis based on age, the results for ampicillin and cefotaxime were mirrored in the pediatric population; however, results were insignificant in the adult population. Subgroup analysis of the adult population showed significance in terms of de-escalation/discontinuation of acyclovir reported (in median hours) as 49 (68) in pre-PCR and 19 (18) in post-PCR group (P = 0.002).ConclusionTime to de-escalation and/or discontinuation of ampicillin and cefotaxime was significantly reduced after implementation of the ME panel suggesting clinical significance in high-risk populations such as neonates. Time to de-escalation and/or discontinuation of acyclovir was significantly reduced in the adult population.Disclosures All authors: No reported disclosures.

The Gene Mutation Spectrum of Breast Cancer Analyzed by Semiconductor Sequencing Platform.

To use the semiconductor sequencing platform (SSP) to analyze the gene mutate spectrum of breast cancer patients. We recruited 46 breast cancer patients, and detected the ER/PR/HER2 expression level of the tumor tissue by immunohistochemistry. In addition, combined with SSP technology, we detected 207 hot mutation regions of 50 breast cancer related genes with multiple polymerase chain reaction (PCR) technology. There were 8 cases of grade I, 18 cases of grade II, 20 cases of grade III in 46 breast cancer patients according to histological grade and 12 cases of ER/PR + HER2 +, 18 cases of ER/PR + HER2-, 13 cases of ER/PR - HER2 +, 20 cases of ER/PR - HER2- according to ER/PR/HER2 status classification. Moreover, we found that there were 33 gene locus mutations of 8 genes including AKT1, APC, BRAF, CDKN2A, KRAS, PTEN, PIK3CA and TP53, but difference was not statistically significant (P > 0.05) when compared these gene mutations (except for PIK3CA) in each groups according to the histological classification of breast cancer and the ER/PR/HER2 classification. PIK3CA mutation rate of grade I was obviously higher than that of grade II ~ III histological grading in breast cancer patients (P < 0.05). Based on our results, we drew a conclusion that the occurrence and development of breast cancer was a process involved multiple genes. Here, we found that PIK3CA played a role in the development of the early stage of breast cancer, which could provide clinical basis for treatment of breast cancer. Moreover, SSP technology could be an effective and sensitive method for detection of gene mutation spectrum in breast cancer.

Archaeal DNA polymerases: new frontiers in DNA replication and repair.

Archaeal DNA polymerases have long been studied due to their superior properties for DNA amplification in the polymerase chain reaction and DNA sequencing technologies. However, a full comprehension of their functions, recruitment and regulation as part of the replisome during genome replication and DNA repair lags behind well-established bacterial and eukaryotic model systems. The archaea are evolutionarily very broad, but many studies in the major model systems of both Crenarchaeota and Euryarchaeota are starting to yield significant increases in understanding of the functions of DNA polymerases in the respective phyla. Recent advances in biochemical approaches and in archaeal genetic models allowing knockout and epitope tagging have led to significant increases in our understanding, including DNA polymerase roles in Okazaki fragment maturation on the lagging strand, towards reconstitution of the replisome itself. Furthermore, poorly characterised DNA polymerase paralogues are finding roles in DNA repair and CRISPR immunity. This review attempts to provide a current update on the roles of archaeal DNA polymerases in both DNA replication and repair, addressing significant questions that remain for this field.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

A0879 Analysis of structural change of gut microbiota in 2K1C hypertensive rats

Objectives: To characterize the gut microbial community structure of two-kidney-one-clip (2K1C) hypertensive rats, providing new ideas for prevention and treatment of hypertension. Methods: The 2K1C hypertensive rat model was established. The total fecal DNA of rats in 2K1C model group and sham operation group were extracted. The structure and principal components of intestinal flora in 2K1C model rats and sham operated rats were then analyzed using Hiseq3000 sequencing platform. Additionally, real time fluorescent quantitative polymerase chain reaction technology was used to detect the content of predominant bacteria, including Escherichia, Bifidobacterium, Lactobacillus and Bacteroides. Results: Compared with sham operation group, significant reduction in the relative abundance of Firmicutes and increases in Bacteroidetes were observed in the fecal samples from model group (P < 0.05). When compared with sham operation group, it also found a significant increase in the relative abundance of Escherichia coli and decrease in the short chain fatty acid producing strains in model group, such as Coprococcus and Roseburia (P < 0.05). Moreover, the relative abundance of Bacteroidetes/Prevotella in model group tended to increase compared with sham operation group (P < 0.05). However, the relative abundance of Firmicutes/Ruminococcus was significant higher than sham operation group (P < 0.05). Principal coordinate analysis showed that there was significant difference in the gut microbial community structure between 2K1C model group and sham operation group. Conclusion: There were significant differences in the gut microbial community structure between 2K1C model group and sham operation group, indicating that gut microbiota may be involved in the pathogenesis of renovascular hypertension in rats.

Analysis of structural features of gut microbiota in two-kidney-one-clip hypertensive rats based on high-throughput sequencing technology

Objective: To characterize the gut microbial community structure of two-kidney-one-clip (2K1C) hypertensive rats, provide new evidences for prevention and treatment of hypertension. Methods: Adult male SD rats were divided into 2K1C hypertensive model group and sham operation group (n=8 each). Blood pressure was recorded by tail-cuff technique weekly for 4 weeks. Then, the total fecal DNA of rats in 2K1C model group and sham operation group were extracted. The structure and principal components of intestinal flora in 2K1C model rats and sham operated rats were analyzed using Hiseq3000 sequencing platform. Additionally, real time fluorescent quantitative polymerase chain reaction technology was used to detect the content of predominant bacteria, including Escherichia, Bifidobacterium, Lactobacillus and Bacteroides. Results: Compared with sham operation group, significant reduction in the relative abundance of Firmicutes (31.72 (30.06, 32.18) vs. 40.99 (38.78, 44.41), U=0.00, P<0.05) and increase in Bacteroidetes (48.13(41.16,50.25) vs. 27.81(25.84,31.38),U=0.00, P<0.05) were observed in the fecal samples from model group. The relative abundance of Escherichia coli (0.08(0.07,0.11) vs. 0.07(0.06,0.08),U=12.50, P<0.05) was increased and the short chain fatty acid producing strains such as Coprococcus, Roseburia, Blautia, Clostridium and Bacteroides was reduced in 2K1C model group than in sham operation group (all P<0.05). Moreover, the relative abundance of Prevotella (Bacteroidetes) was also significantly higher in 2K1C model group than in sham operation group (18.14(17.78,18.75) vs.1.83(1.50,5.19), U=0.00, P<0.05). Moreover, the relative abundance of Ruminococcus (Firmicutes) was significant higher in 2K1C model group than in sham operation group (7.73(6.04,9.34) vs. 3.68(2.46,4.67),U=0.00, P<0.05). Principal coordinate analysis showed that there was significant difference in the gut microbial community structure between 2K1C model group and sham operation group. Conclusion: There are significant changes in the gut microbial community structure in 2K1C model group as compared to sham operation group, indicating renovascular hypertension might affect gut microbial community structure in this rat model.

Identification of key tumorigenesis‑related genes and their microRNAs in colon cancer.

Colorectal cancer(CRC) is one of the most common malignant tumors and its development involves multi‑gene driven processes that affect the digestive system. The objective of this study was to identify tumorigenesis‑associated gene signatures using microarray expression profiling data. The gene expression profiling of GSE39582, a dataset containing 566colon cancer samples and 19non‑tumoral colorectal mucosae was downloaded from Gene Expression Omnibus (GEO) database. A total of 439differentially expressed genes (DEGs) were extracted by GEO2R. Many of these DEGs were cancer‑related, involved in the regulation of cell proliferation, extracellular matrix (ECM)‑receptor interaction and phosphatidylinositol3‑kinase(PI3K)‑Akt signaling pathway according to the results of pathway enrichment analysis in Database for Annotation, Visualization and Integrated Discovery(DAVID). Then, 10genes were predicted to play an important role in the development of CRC. Transient receptor potential cation channel, subfamilyM, and member6 (TRPM6), a member of 10hub genes, was confirmed to be downregulated in 16(80%) of 20colon cancer tissues using quantitative polymerase chain reaction(qPCR) technology. Furthermore, high expression of TRPM6 was indicative of a prolonged overall survival(OS) in CRC patients through the analysis of GSE39582. Hsa‑let‑7g and hsa‑let‑7f‑1 were believed to be the regulatory miRNAs of TRPM6 by TCGA and miRanda database. In conclusion, this study may play a critical role in promoting the discovery of potential targets for diagnosis, treatment and prognosis of CRC.

Development of a DNA methylation-based semen-specific SNP typing method: A new approach for genotyping from a mixture of body fluids

Genotyping from samples containing different types of body fluids is a major difficulty in forensic investigations. Recently, CpG sites that are specifically methylated or unmethylated in different types of body fluids have been reported as novel markers for body fluid identification. In this study, we hypothesized that the simultaneous analysis of CpGs and neighboring polymorphic sites on the same molecule could be useful for individual DNA typing from mixed samples. We performed a proof-of-concept study of this approach by searching the genome-wide methylation dataset deposited at the National Center for Biotechnology Information Gene Expression Omnibus repository for semen-specific CpG markers adjacent to common single nucleotide polymorphisms. From the identified candidates, we selected 5 regions on different chromosomes and validated the presence of semen-specific methylation or unmethylation in each region by pyrosequencing analyses. By combining methylation-specific polymerase chain reaction and pyrosequencing technology, we developed a semen-specific DNA typing method for two semen-specific methylated regions and one semen-specific unmethylated region. Finally, the method successfully identified semen-derived alleles from mixed stains, indicating that this methylation-based approach can be applicable to actual forensic samples. Since existing separation techniques physically isolate cells derived from each type of body fluid, this approach may be useful when existing methods cannot be performed due to the degradation of samples.

Association between the methylation of six apoptosis‑associated genes with autism spectrum disorder.

Excessive apoptosis hinders the process of brain maturation and is regarded as one of the principal risk factors for the development of autism spectrum disorder (ASD). The aim of the present study was to investigate the association between the methylation of six apoptosis‑associated genes [transforming growth factor β 1 (TGFB1), BCL2 associated X, apoptosis regulator, insulin like growth factor binding protein 3, protein kinase C β 1, presenilin 2 and C‑C motif chemokine ligand 2] and ASD. Using quantitative methylation‑specific polymerase chain reaction technology, DNA methylation levels were detected in 42 autistic and 26 control subjects. The logistic regression analysis results demonstrated that of the six genes, only TGFB1 was significantly hypomethylated in peripheral blood samples from children with autism compared with control samples (mean percentage of methylated reference, 0.011% vs. 0.019%; age‑adjusted P=0.028). In addition, TGFB1 methylation was identified to be positively associated with the interaction ability score from the Autism Behavior Checklist (r=0.452; P=0.035). These data suggested that decreased TGFB1 methylation may contribute to the development of ASD.

Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids.

Plant viruses are still one of the main contributors to economic losses in agriculture. It has been estimated that plant viruses can cause as much as 50 billion euros loss worldwide, per year. This situation may be worsened by recent climate change events and the associated changes in disease epidemiology. Reliable and early detection methods are still one of the main and most effective actions to develop control strategies for plant viral diseases. During the last years, considerable progress has been made to develop tools with high specificity and low detection limits for use in the detection of these plant pathogens. Time and cost reductions have been some of the main objectives pursued during the last few years as these increase their feasibility for routine use. Among other strategies, these objectives can be achieved by the simultaneous detection and (or) identification of several viruses in a single assay. Nucleic acid-based detection techniques are especially suitable for this purpose. Polyvalent detection has allowed the detection of multiple plant viruses at the genus level. Multiplexing RT polymerase chain reaction (PCR) has been optimized for the simultaneous detection of more than 10 plant viruses/viroids. In this short review, we provide an update on the progress made during the last decade on techniques such as multiplex PCR, polyvalent PCR, non-isotopic molecular hybridization techniques, real-time PCR, and array technologies to allow simultaneous detection of multiple plant viruses. Also, the potential and benefits of the powerful new technique of deep sequencing/next-generation sequencing are described.

Use of plasma mitochondrial DNA levels for determining disease severity and prognosis in pediatric sepsis: a case control study

BackgroundThe mortality rate due to severe sepsis is approximately 30–60%. Sepsis readily progresses to septic shock and multiple organ dysfunction, representing a significant problem in the pediatric intensive care unit (PICU). The aim of this study was to explore the value of plasma mitochondrial DNA (mtDNA) for early diagnosis and prognosis in children with sepsis.MethodsA total of 123 children with sepsis who were hospitalized in the Hunan Children’s Hospital PICU from July 2013 to December 2014 were divided into the general sepsis group (n = 70) and severe sepsis group (n = 53) based on diagnostic standards. An additional 30 children with non-sepsis infection and 30 healthy children were randomly selected as a control group. Patients’ plasma was collected during admission to the PICU. A pediatric critical illness score (PCIS) was also calculated. The plasma mtDNA level was examined using real-time polymerase chain reaction technology, and other parameters including routine laboratory values; blood lactate, procalcitonin (PCT), and C-reactive protein (CRP) levels; and data on survival were collected and compared among the groups.ResultsThe plasma mtDNA level in the sepsis group than that in the non-sepsis infection and healthy groups. The plasma mtDNA level was significantly higher in the severe sepsis than in the general sepsis group (p < 0.001). A lower PCIS was associated with a higher plasma mtDNA level (p < 0.001). A higher number of organs with dysfunction was associated with higher plasma mtDNA levels (p < 0.001). Plasma mtDNA levels were higher among patients with elevated alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, lactate dehydrogenase, creatine kinase, myoglobin, creatine kinase MB, and troponin than in those with values within the normal range. The mtDNA level was higher among non-survivors than among survivors, and this difference was significant. mtDNA showed a prognostic prediction value similar to that of lactate, PCT, and CRP.ConclusionsPlasma mtDNA levels may be a suitable biomarker for diagnosis and prognosis in children with sepsis.

Cell-free RNA studies in cancer based on T oligo-primed polymerase chain reaction (TOP-PCR) technology

Cell-free RNA studies in cancer based on T oligo-primed polymerase chain reaction (TOP-PCR) technology