PCR Capillary Electrophoresis Research Articles

Clinicopathological features and types of microsatellite instability in 1394 patients with colorectal cancer

To explore the clinicopathological features and types of genic mutations in DNA mismatch repair (MMR) in colorectal cancer (CRC). Immunohistochemistry was used to determine the expression of MMR proteins in 1394 patients with CRC, and PCR-capillary electrophoresis (PCR-CE) was used to detect microsatellite instability (MSI) in 106 cases of defective MMR (dMMR), 46 cases of proficient MMR (pMMR) with heterogeneous expression and 147 randomly selected cases of pMMR. The relationship between the expressions of MMR proteins and the clinicopathological features of the patients was evaluated. The consistency between the results of immunohistochemistry and PCR-CE was assessed. Immunohistochemical staining showed an incidence of dMMR of 7.6% in the patients. The main type of dMMR was co-deletion of MLH1 and PMS2, accounting for 55.7% of the total dMMR cases. The deletion of MMR proteins was significantly correlated with the patients' age, tumor location, tumor size, gross type, histological type, degree of differentiation, lymph node status and TNM stage (P < 0.05), but not with gender (P > 0.05). The total accordance rate of immunohistochemistry and PCR-CE was 98.7% in these patients. The main type of dMMR is co-deletion of MLH1 and PMS2 in patients with colorectal cancer. dMMR colorectal cancer has typical clinicopathological features and a lower incidence in China than in Western countries. The results of immunohistochemistry and PCR-CE are highly consistent for detecting dMMR in colorectal cancer patients.

Association of TNF rs668920841 and INRA111 polymorphisms with caprine brucellosis: A case-control study of candidate genes involved in innate immunity

Caprine brucellosis is an infectious, contagious zoonotic disease caused by Brucella melitensis. Multiple factors, including host genetics, can influence the outcome of the exposure to Brucella; and it is expected that genetic variants that affect the host innate immune response could have a key role in Brucella infection and pathogenesis. In this study, we evaluated if polymorphisms in innate immunity-related genes are associated with results of Brucella infection in goats. Nine polymorphisms within interferon gamma (IFNG), tumor necrosis factor (TNF), MyD88 innate immune signal transduction adaptor (MYD88), interleukin 10 (IL10) and IL-10 receptor subunit alpha (IL10RA) genes and two molecular markers (BMS2753 and INRA111) were resolved by PCR-capillary electrophoresis in samples from 81 seronegative and 61 seropositive goats for brucellosis. A heterozygous genotype at INRA111, a microsatellite near the VRK serine/threonine kinase 2 (VRK2) gene, was associated with absence of Brucella-specific antibodies in goats naturally exposed to the pathogen (P = .004). Conversely, variants in the TNF gene (rs668920841) and near the IFN gamma receptor 1 (IFNGR1) gene (microsatellite BMS2753) were significantly associated with presence of Brucella-specific antibodies at allelic (P = .042 and P = .046) and genotypic level (P = .012 and P = .041, respectively). Moreover, an in silico analysis predicted a functional role of the insertion-deletion polymorphism rs668920841 on the transcriptional regulation of the caprine TNF gene. Altogether, these results contribute to the identification of genetic factors that have a putative effect on the resistance / susceptibility phenotype of goats to Brucella infection.

A barcode-DNA analysis method for the identification of plant oil adulteration in milk and dairy products

In the present work, a barcode-DNA analysis method is described for the detection of plant oil adulteration in milk and dairy products. The method relies on the fact that plant DNA should not be present in readily detectable amounts in a dairy product unless it contains undeclared plant material. Thus, a universal plant barcode is chosen as the target to be amplified from dairy samples. Accordingly, barcode PCR-CE (PCR-capillary electrophoresis) assays are described, which do not require preliminary information on the species source of the adulterant oil type. Two PCR-CE assays, one operating on the plastid trnL (UAA) intron and the other targeting its inner P6 loop in nested format, were shown to detect corn, soybean, rapeseed and sunflower oils in clarified butter, milk and yogurt. Both barcodes are robustly amplified with extremely conserved primers. While the intron provides the species discrimination ability, the P6 loop provides superior detection sensitivity.

Cytopathological tests for early detection of oral carcinogenesis.

The carcinogenesis in the oral cavity occurs as a multistep process and is often preceded by potentially malignant lesions. The main risk factors for the development of oral cancer are smoking and alcohol intake. The current challenge is to identify patients at greatest risk for the development of oral cancer using noninvasive and effective methods. The aim of this study is to evaluate the microsatellite mutations in the 9p21 locus, the cell proliferative activity, the pattern of epithelial desquamation, and the nucleus/cytoplasm ratio of exfoliated epithelial cells. Cytopathological samples were collected from 131 individuals divided into four groups: control (n = 26), alcohol-smoking (n = 32), leukoplakia (n = 38), and the oral squamous cell carcinoma group (OSCC, n = 35). From the cytological scraping, a slide was silver impregnated for Ag-stained nucleolar organizer region analysis and another slide was stained using the Papanicolaou technique. The remaining cells were used for DNA extraction, followed by PCR amplification and capillary electrophoresis. The cell proliferation velocity rate was higher in the leukoplakia and OSCC groups compared with the control group (P < 0.05). The leukoplakia group showed increased anucleated scales, whereas the nucleated superficial predominated in the control group and the parabasal cells in the OSCC group (P < 0.05). An increased nucleus/cytoplasm ratio was detected only in the OSCC group (P < 0.05). The 9p21 locus mutation frequency was higher in the alcohol-smoking and leukoplakia groups. 9p21 analysis and Ag-stained nucleolar organizer region methods are promising for the screening and monitoring of individuals at higher risk for the development of oral cancer.

Identification of diarrheagenic Escherichia coli by a new multiplex PCR assay and capillary electrophoresis

Diarrheagenic Escherichia coli (DEC) is a set of the most common pathogens causing diarrhea. DEC strains are classified into five pathotypes based on the possession of different virulence genes: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). The development of an easy-to-use method to detect the specific virulence genes and distinguish the pathotypes is essential for the diagnosis and surveillance of DEC infections. In this study, a multiplex PCR assay (mPCR) specific to nine virulence genes and an internal control was designed for the identification of five DEC pathotypes. A temperature switch PCR (TSP) strategy was used in the PCR amplification. The PCR products were detected by capillary electrophoresis. The limit of detection (LOD) of the 10-plex reaction was 5 × 103 copies/reaction for stx2 and 5 × 102 copies/reaction for the other targets. The mPCR showed very high specificity, and inclusivity and exclusivity were both 100%. When the mPCR assay was used for the detection of 221 cryopreserved diarrhea specimens, DEC colonies were detected from 49 specimens, and the positive rate was 22.2%. The mPCR assay was sensitive and specific, and the amplified product could be analyzed easily. Thus, this method could be used effectively to identify the suspected colonies of DEC in the primary culture of the specimen.

Evaluation of 30 insertion/deletion polymorphisms as forensic markers in the Kuwaiti population

Insertion/deletion (indel) polymorphism markers have been developed as a supplementary genotyping system in forensic DNA testing. The only commercial indel genotyping kit is the Qiagen Investigator® DIPplex kit, which includes 30 loci that can be typed using PCR-capillary electrophoresis (PCR-CE). We show that these 30 markers can be used to genetically assign Kuwaiti individuals to their population of origin when compared to other distant populations, using principal components analysis (PCA) and STRUCTURE. We also demonstrate a relationship between geographic and genetic distance using allele frequencies for the 30 markers from 26 populations.

Expansion of trinucleotide CTG repeats in the TCF4 gene as a marker of fuchs’ endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is an inherited severe and progressive disease, characterized by endothelial cell density decrease and increasing corneal edema. FECD development may be linked to expanded trinucleotide repeat, CTG, in the third intron of the TCF4 gene. The study focuses on estimating the prevalence of expanded CTG repeat in TCF4 gene in the Russian population, in patients with normal cornea and in patients with FECD (by applying triplet repeat PCR technique and capillary electrophoresis). 51 patients with FECD and 38 patients with normal cornea were examined. The estimation of the number of CTG triplet repeats in TCF4 gene determination is the veracious laboratory marker of FECD.

Validation of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing.

e13138 Background: Validation is very important for diagnostic assays, here we describe a validation process of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing. This assay can detect base substitutions, short insertions and deletions, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI) across 543 cancer-related genes from tumor specimens or plasma. Methods: Analytical validation was conducted with 20 cell line pools whose mutation were verified by digital droplet PCR (ddPCR). Clinical validation was conducted with Proficiency Test samples and clinical samples whose mutation were verified by ddPCR. Sensitivity, positive predictive value (PPV) and precision of tumor specimens and plasma were assessed across the reportable range of the assay. For measurement of TMB， NGS libraries of a cohort of tumor samples were tested by whole exosome panel (WES) and this assay respectively. And clinical tumor tissue samples whose MSI status were identified by fluorescent PCR-capillary electrophoresis were also tested by this assay. Results: The SNV/Indel LOD95 was 0.6% for hot-spot variants, and 1.3% for panel-wised variants of tumor tissue samples with high specificity (PPV &gt;99%). The SNV/Indel LOD95 was 0.4% for hot-spot variants, and 1.1% for panel-wised variants of plasma samples (cfDNA≥15ng), with PPV &gt;99%. LOD95 of CNV was 2.2-2.3, with PPV&gt;99%. Precision of CNV was higher than 95%, precision of SNV/Indel was higher than 97%. Concordance between TMB results tested by WES and this assay was 0.94. MSI results identified by this assay was the same as fluorescent PCR-capillary electrophoresis method. Conclusions: In summary, we present the analytical and clinical validation of a comprehensive NGS-based diagnostic assay for comprehensive tumor genomic profiling.

Association of an IRF3 putative functional uORF variant with resistance to Brucella infection: A candidate gene based analysis of InDel polymorphisms in goats

Brucellosis is an important zoonotic disease caused by infection with Brucella spp. It generates major economic losses in livestock production worldwide. Goats are the principal hosts of B. melitensis, the main infection agent of caprine and human brucellosis. The selection of resistance-related genes is considered one of the best long-term means to improve control to bacterial infection in domestic ruminants. We performed a candidate gene association study to test if six short insertion/deletion polymorphisms (InDels) at bacterial-infection related genes influence the resistance to Brucella infection in female creole goats. InDels (IRF3-540: rs660531540, FKBP5-294: rs448529294, TIRAP-561: rs657494561, PTPRT-588: rs667380588, KALRN-989: rs667660989 and RAB5a-016: rs661537016) were resolved by PCR-capillary electrophoresis in samples from 64 cases and 64 controls for brucellosis. Allelic frequencies were significantly different between cases and controls at IRF3-540 and KALRN-989 (p = 0.001 and 0.005). Indeed, the minor alleles (a and k) at InDels IRF3-540 and KALRN-989 were more frequent among controls than cases, providing evidence that these alleles confer protection against Brucella infection. Moreover, IRF3-540 a-containing genotypes (Aa and aa) were associated with absence of Brucella-specific antibodies in goats (p = 0.003; OR = 3.52; 95% CI = 1.55–7.96), and more specifically, a-allele was associated with resistance to Brucella infection in a dose-dependent manner. Also, we observed that the IRF3-540 deletion (a-allele) extends a conserved upstream ORF by 75 nucleotides to the main ORF, and thus it may decrease gene expression by reducing translation efficiency from the main ORF. These results suggest a potential functional role of IRF3-540 deletion in genetic resistance to Brucella infection in goats.

Plastid trnH-psbA intergenic spacer serves as a PCR-based marker to detect common grain adulterants of coffee (Coffea arabica L.)

The present work describes the utilization of interspecific length polymorphisms within the chloroplastic trnH-psbA intergenic spacer to standardize a DNA-based method that identifies adulteration in Arabica coffee (Coffea arabica L.). Adulteration of coffee with roasted cereal grains and soybean is a common fraudulent practice, yet, research to design sensitive DNA-based tests in order to establish traceability in coffee is extremely limited. In the present study, PCR-CE (PCR-capillary electrophoresis) analysis of the trnH-psbA intergenic spacer proved successful to discriminate Arabica coffee and its common adulterants, namely corn, soybean, rice, wheat and barley. When the barcode PCR-CE assay was tested on admixtures, adulteration as low as 1% was successfully detected simply through barcode amplification and capillary electrophoresis separation. Moreover, it is important to note that the proposed barcode assay not only detects adulteration but also reveals the identity of the adulterant species based on barcode amplification profiles obtained from reference sets of anticipated adulterants.

Drowning Diagnosis by Detecting Diatoms rbcL Genes with PCR-Capillary Electrophoresis

Drowning Diagnosis by Detecting Diatoms<i> rbc</i>L Genes with PCR-Capillary Electrophoresis

Characterizing Cell Heterogeneity Using PCR Fingerprinting of Surface Multigene Families in Protozoan Parasites.

Parasites counteract the action of the immune system and other environmental pressures by modulating and changing the composition of their cell surfaces. Surface multigene protein families are defined not only by highly variable regions in length and/or sequence exposed to the outer space but also by conserved sequences codifying for the signal peptide, hydrophobic C-terminal regions necessary for GPI modifications, as well as conserved UTR regions for mRNA regulation. The method here presented exploits these conserved signatures for characterizing variations in the mRNA expression of clonal cell populations of protozoan parasites using a combination of nested PCR amplification and capillary electrophoresis. With this workflow, in silico gels from isolated cell clones can be generated, thus providing an excellent tool for analyzing cellular heterogeneity in protozoan parasites.

A simple microsatellite-based method for hazelnut oil DNA analysis

Molecular food traceability requires continuous updates to identify more robust, efficient and affordable methodologies to guarantee food quality and safety and especially consumers’ health. Available commercial kits are often unsatisfactory and require modifications to successfully detect single components on complex and transformed food matrices. Here we report a simple method for molecular traceability of cold-pressed hazelnut oil based on microsatellite DNA markers. Different genomic extraction methodologies were tested and a total genome pre-amplification step was applied on PCR-negative samples. PCR-capillary electrophoresis using nine microsatellites demonstrates the accuracy of the fingerprint analysis even for filtered oil.

The Importance of a Comparative Characterization of Saccharomyces Cerevisiae and Saccharomyces Pastorianus Strains for Brewing

The volume and market share loss for classical beer types such as pils beer and wheat beer has been declining for several years, but the overall beer market remains almost unchanged as a result of the increasing interest in beer specialties Due to high biodiversity, the diversity of the strains, and the different flavor profiles, reliable and practical information regarding the characteristics of individual brewing strains is required to help brewers to find the right strain for their brewing purposes. This paper presents a comparison of 10 commercially available Technical University of Munich (TUM) brewing yeast strains. The strains were screened for genetic and phenotypic characteristics. After confirming the genetic distinctiveness by using species-specific real-time polymerase chain reaction (RT-PCR) systems and a strain typing method based on PCR-capillary electrophoresis of the partial intergenic spacer 2 (IGS2) fragment (IGS2-314 PCR-capillary electrophoresis), the strains were tested regarding phenotypic characteristics under controlled and identical fermentation conditions in small-scale brewing trials. Besides the fermentation performance, flocculation behavior, sugar metabolism and other phenotypic characteristics, the main focus was on the flavor and aroma profile of each investigated TUM yeast strain.

Effects of VKORC1 and CYP2C9 gene polymorphisms on the stable dosage of warfarin and anticoagulation in patients with hypertrophic cardiomyopathy complicated with atrial fibrillation

Objective To investigate the gene polymorphisms and clinical features of vitamin K epoxide reductase complex 1(VKORC1) and cytochrome P450 2C9(CYP2C9), and their correlation with stable dosage of warfarin, the risk of hemorrhage and over anticoagulation for patients with hypertrophic cardiomyopathy. Methods A retrospective study was performed on 60 patients with hypertrophic cardiomyopathy(HCM) combined with atrial fibrillation(AF) who were admitted and regularly took oral warfarin as anticoagulation from January 2011 to December 2016.The clinical data such as age, body height, body weight, stable warfarin dosage and the situation of hemorrhage and over anticoagulation were recorded.Genotypes of CYP2C9 and VKORC1 were detected with fluorescence PCR-capillary electrophoresis sequencing and the relationship between the genotype and clinical features and the stable dose of warfarin, hemorrhage and over anticoagulation were analyzed by statistical methods. Results Among the 60 patients, VKORC1-1639G>A detection showed that 50 patients were AA type, 9 patients were AG type, 1 patient was GG type, the stabledosage of AA type patients were significant lower than that of AG type patients(P 0.05); CYP2C9 detection showed that 51 patients were *1/*1 type, 6 patients were *1/*3 type, 3 patients were *3/*3 type, *2 allele was not found, the stable dosage of patients who carry the *3 mutation were significant lower than that of *1/*1 type patients, and the more *3 mutation they own, the lower dosage they require(P 0.05). The incidence of hemorrhage and anticoagulation was significantly higher in patients aged 65 years or older than those who were younger than 65 years(P<0.05). Conclusion Stable warfarin dosage has relationship with different genotypes of VKORC1 and CYP2C9 in HCM patients, age may be a factor of warfarin over anticoagulation and hemorrhage. Key words: Hypertrophic cardiomyopathy; Atrial fibrillation; Warfarin; Vitamin K epoxide reductase complex 1; cytochrome P450 2C9; Gene polymorphisms; Hemorrhage; Over anticoagulation

Establishment and Application of PCR Capillary Electrophoresis System by Detecting the Aeromonas hydrophila Hemolytic Genes for Drowning Diagnosis

Establishment and Application of PCR Capillary Electrophoresis System by Detecting the <i>Aeromonas hydrophila</i> Hemolytic Genes for Drowning Diagnosis

Try to Apply T Cell Receptor Repertoire Diversity Detection for Acquired Immune Deficiency Syndrome Research of Traditional Chinese Medicine

A new approach will be carried out to make clear the mechanism of promotion immune system function with TCM treatment on AIDS and provide a parameter of clinical therapeutic effects. DNA was extracted with periphery blood from forty AIDS patients before and after treatment, and multiple primers PCR technique and capillary electrophoresis were used to detect T cell receptor beta repertoire diversity. Meanwhile, seven control subjects were measured repeatedly to define the threshold. Sets of typical patterns for positive and negative were shown respectively from the scanning map angle. Moreover, positive detection rates of the forty AIDS patients were calculated for different T cell receptor gene segments. To some degree, this study is suggestive of it how TCM treatment effects on AIDS. However, the reliability needs further verification.

16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

Alternative splicing of SMPD1 coding for acid sphingomyelinase in major depression

BackgroundMajor depressive disorder (MDD) is a psychiatric disorder characterized by key symptoms that include depressed mood and a loss of interest and pleasure. A recently developed pathogenic model of MDD involves disturbed neurogenesis in the hippocampus, where the acid sphingomyelinase (ASM)/ceramide system plays an important role and is proposed as a molecular target for antidepressant action. Because alternative splicing of SMPD1 mRNA, coding for ASM, is relevant for the regulation of ASM enzymatic activity, we investigated the frequency of alternatively spliced ASM isoforms in peripheral blood cells of MDD patients versus healthy controls. MethodsBecause the full-length transcript variant 1 of SMPD1 (termed ASM-1) is the only known form within the splicing pattern that encodes an enzymatically fully active ASM, we determined a fraction of splice isoforms deviating from ASM-1 using PCR amplification and capillary electrophoresis with laser-induced fluorescence analysis. ResultsASM alternative splicing events occurred significantly less frequently in MDD patients compared to healthy subjects. After 5 days of antidepressant treatment, the frequency of alternatively spliced ASM isoforms decreased in those patients who were treated with a functional inhibitor of ASM activity (FIASMA) but remained constant in MDD patients treated with other antidepressant drugs. This effect was more pronounced when healthy male volunteers were treated with the FIASMAs fluoxetine or paroxetine, in contrast to a placebo group. LimitationsPatients were treated with different antidepressant drugs, depending on individual parameters and disease courses. ConclusionsThis study shows that the ASM alternative splicing pattern could be a biological target with diagnostic relevance and could serve as a novel biomarker for MDD.

Regulation of alternative splicing in human obesity loci.

Multiple obesity susceptibility loci have been identified by genome-wide association studies, yet the mechanisms by which these loci influence obesity remain unclear. Alternative splicing could contribute to obesity by regulating the transcriptomic and proteomic diversity of genes in these loci. Based on a database search, 72 of the 136 genes at the 13 obesity loci encoded multiple protein isoforms. Thus, alternative splicing of these genes in adipose tissue samples was analyzed from the Metabolic Syndrome in Men population-based study and from two weight loss intervention studies (surgical and very low calorie diet). Alternative splicing was confirmed in 11 genes with PCR capillary electrophoresis in human subcutaneous adipose tissue. Interestingly, differential splicing of TRA2B, BAG6, and MSH5 was observed between lean individuals with normoglycemia and overweight individuals with type 2 diabetes. Of these genes, we detected fat depot-dependent splicing of TRA2B and BAG6 and weight loss-induced regulation of MSH5 splicing in the intervention studies. Finally, body mass index was a major determinant of TRA2B, BAG6, and MSH5 splicing in the combined data. This study provides evidence for alternative splicing in obesity loci, suggesting that alternative splicing at least in part mediates the obesity-associated risk in these loci.