SYBR Green-based Real-time Polymerase Chain Research Articles

Plasma cell-free DNA integrity index and hepatocellular carcinoma treated or not with direct-acting antivirals: A case-control study

Background and study aimsThe clinical value of the cell-free DNA (cf-DNA) integrity index as a diagnostic biomarker of hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) was investigated and correlated with alpha-fetoprotein (AFP). Patients and methodsThis case-control study was conducted on 160 patients with HCV genotype 4-related liver cirrhosis. Group 1 consisted of 80 patients with HCC, including 40 patients naïve to direct-acting antivirals (DAAs) and 40 patients who received DAAs and achieved sustained virological response. Group 2 comprised 80 patients with cirrhosis without HCC. Plasma cf-DNA integrity index using ALU 115 and ALU 247 sequences was assessed using SYBR Green-based real-time polymerase chain reaction (RT-PCR). The cf-DNA integrity index was calculated as the ratio of Q247/Q115 where Q115 and Q247 are the ALU-qPCR results obtained using ALU 115 and ALU 247, respectively. ResultsPatients with HCC had significantly lower plasma cf-DNA integrity index than those with liver cirrhosis. No significant difference in the cf-DNA integrity index was observed between patients with HCC who received DAAs and those who did not. Receiver operating characteristic (ROC) analysis revealed an area under the ROC curve of 0.965 and 0.886 for detecting HCC using the cf-DNA integrity index and AFP, respectively. The combination of the cf-DNA integrity index and AFP improved the sensitivity from 81.6% to 94.7%, positive predictive value from 93.4% to 94.7%, negative predictive value from 84.4% to 94.9%, and accuracy from 88.4% to 94.8%. ConclusionThe cf-DNA integrity index can predict the occurrence of HCV genotype 4-related HCC. No significant difference in the cf-DNA integrity index was observed between patients with HCC who received DAAs and those without previous DAAs. The combination of the cf-DNA integrity index and AFP provides better HCC prediction accuracy.

Development of Real-Time PCR Based on A137R Gene for the Detection of African Swine Fever Virus

African swine fever virus (ASFV) can infect domestic pigs and wild boars and causes huge economic losses in global swine industry. Therefore, early diagnosis of ASFV is important for the control and eradication of African swine fever (ASF). In this study, a SYBR Green-based real-time polymerase chain reaction (PCR) assay targeting the viral encoded A137R gene was established for the detection of ASFV infection. For the evaluation of the established real-time PCR, 34 clinical samples were assessed by both the A137R gene-based real-time PCR and OIE-recommended TaqMan PCR. The results showed that 85.29% (29/34) were detected by A137R gene-based real-time PCR, but only 79.41% (27/34) positive using OIE-recommended TaqMan PCR. Moreover, no cross-reaction with other common swine pathogens was found in the A137R gene-based real-time PCR. These results demonstrated that the established real-time PCR assay in this study showed better performance than the OIE-recommended method in detecting ASFV from clinical samples, which could be applied for control and eradication programs of ASF.

Genetic Markers Indicate that 1,25-dihydroxyvitamin D Treatment may not Protect Against Hepatocellular Carcinoma

Objectives: The impact of 1,25-dihydroxyvitamin D on hepatocellular carcinoma (HCC) cells is a complicated area. In this study, we aimed to clarify the effect of 1,25-dihydroxyvitamin D on HCC cells according to genetic markers. Methods: The optimal concentration of 1,25-dihydroxyvitamin D is treated to HepG2 cells (250 nM at the 48th hour). From treated HepG2 cells, total Ribonucleic Acid (RNA) was isolated, and Ki-67, MMP-2, MMP-9, HIF-1?, hTERT, and piR823 gene expressions were determined by SYBR Green-based real-time polymerase chain reaction. Results: : Increased expressions of Ki-67, hTERT, and piR-823 were determined compared with the control group at the 48th hour after treatment (p<0.001), while decreased gene expressions of MMP-2, MMP-9, and HIF-1? were observed compared with the control group (p<0.001). Conclusion: Currently, there are several different opinions about the usage of vitamin D, especially in HCC. In addition to researchers who argue that vitamin D has anticarcinogenic and protective properties, an increasing number of researchers argue that tumor cells can become aggressive after HCC occurs. According to our results, it was determined that vitamin D causes HepG2 HCC cells to become aggressive in terms of gene expression in the parameters used as a marker for proliferation, adhesion, and differentiation. Keywords: 1.25-Dihydroxyvitamin D, hepatocellular carcinoma, motility, PIWI interacting RNA, proliferation

Development of a recombinase-aided amplification assay for detection of orf virus

Orf, caused by orf virus (ORFV), is an important zoonotic disease that infects goat and sheep, leading to huge economic losses. ORFV can also cause cutaneous lesions in people who come in close contact with the diseased animals. Although accurate diagnostic methods for ORFV infection exist, there is a need for a rapid, specific, and sensitive method for easy clinical application. Here, we successfully established a recombinase-aided amplification (RAA) assay for rapid detection of ORFV. The analytical sensitivity of the assay for ORFV detection is 1 × 101 copies per reaction. Moreover, no cross-reaction was observed with other common DNA viruses. A total of 45 archived suspected ORFV infected nasal scab skin samples were examined by RAA and SYBR Green-based real-time polymerase chain reaction (PCR). Compared with the real-time PCR assay, the kappa values of the RAA assay for ORFV detection was 0.845 (p <0.001), indicating that both assay results were fully in agreement. In conclusion, this detection assay provides a rapid, sensitive, and specific method for ORFV detection and is suitable for ORFV clinical testing.

SYBR Green-based real-time polymerase chain reaction assay for detection of porcine parvovirus 6 in pigs.

In this study, a SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assay was developed for rapid detection of porcine parvovirus (PPV) 6. Primer pairs targeting the conserved regions of PPV6 Capsid gene were designed. Sensitivity analyses revealed the lowest detection limit of the SYBR Green-based real-time PCR assay to be 47.8 copies/μL, which indicated it was 1000 times higher than that found in the conventional PCR investigations. This assay was specific and showed no cross-species amplification with other six porcine viruses. The assay demonstrated high repeatability and reproducibility; the intra- and inter-assay coefficients of variation were 0.79% and 0.42%, respectively. The positive detection rates of 180 clinical samples with SYBR Green-based real-time PCR and conventional PCR were 12.22% (22/180) and 4.44% (8/180), respectively. Our method is sensitive, specific, and reproducible. The use of SYBR Green-based real-time PCR may be suitable for the clinical detection and epidemiological investigation of PPV6.

Development and application of a SYBR green real-time PCR for detection of the emerging avian leukosis virus subgroup K

ABSTRACTAvian leukosis virus subgroup K (ALV-K) is an emerging ALV tumor virus of chickens. We developed a SYBR green-based real-time polymerase chain reaction (PCR) assay for the rapid and economical detection of ALV-K in chicken flocks. The assay was specific for ALV-K and did not cross-react with other ALV subgroup or avian influenza virus, Newcastle disease virus, or Marek's Disease virus. The method was 100 times more sensitive than conventional PCR and 10 times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for the P27 antigen. The assay was also more sensitive than conventional PCR in tests of 86 clinical plasma samples. DF-1 tissue culture cells infected with 1 TCID50 ALV-K particle were identified as negative using ELISA but tested positive with the real-time PCR method. The viral loads in organs and tissues in infected chickens were highest in kidney, lungs, and glandular stomach, and these results matched ELISA findings.

Ungulate copiparvovirus 2 in healthy and postweaning multisystemic wasting syndrome-affected pigs.

A SYBR Green-based real-time polymerase chain reaction (qPCR) was designed to detect Ungulate copiparvovirus 2, also known as porcine parvovirus 4 (PPV4). The test was applied to search for PPV4 DNAemia in sera from 1- to 4-month-old pigs displaying signs of postweaning multisystemic wasting syndrome (PMWS), as well as in sera from healthy swine at equivalent age and in sera from older healthy animals (>6months old). High levels of PPV4 DNA were detected in PMWS-affected pigs. The mean viral DNA load in PMWS-affected pigs was 5.2×107 copies/mL, whereas in young healthy pigs it was 1.4×105 copies/mL (P≤0.001). Although the copy numbers were lower in younger PMWS-affected individuals, this result sheds some light on the possible association between PPV4 viral load detection in this group and the immune impairment caused by PMWS.

Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka

Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc – MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.

Advancements in diagnosis and control measures of viral pathogens in aquaculture: an Indian perspective

Indian aquaculture industry was growing steadily and showed a sixfold growth in production over the last two decades. However, the overall development of aquaculture in India did not reach the levels as projected due to frequent disease outbreaks and related issues. Shrimp and fish are predominantly affected by viruses including white spot syndrome virus, monodon baculovirus, hepatopancreatic parvovirus, viral nervous necrosis, white tail disease, and hypodermal and hematopoietic necrosis baculovirus that cause outbreaks across the countries. Owing to these viral pathogens, the production of aquaculture fishes and crustaceans has dramatically been dropped. There are no specific measures to control these viral infections since it causes mortality at all life stages of cultured aquatic organisms. Early detection of the diseases may be beneficial to prevent the spreading and mass mortality. In India, the Central Institute of Brackish Water Aquaculture has developed a non-invasive diagnostic tool for early and precise detection of monodon baculovirus infection in Indian tiger shrimp Penaeus monodon using SYBR Green-based real-time polymerase chain reaction (PCR) technique. More than twenty-five cell lines from freshwater, brackish water, and marine fish have been developed, characterized, and stored in C. Abdul Hakeem College for research and viral diagnosis. This review reveals the distinctive tools that are being used in aquaculture for the detection of pathogens and preventive measures. Advanced molecular methods such as nested PCR and SYBR Green-based real-time PCR are found to be sensitive and effective for the quantitation. Simple and inexpensive methods such as microscopic evaluation and histopathological analysis detects the progression of the disease. These assays can be used as a diagnostic method for emerging diseases in addition to avoid forthcoming of another.

Enteroaggregative Escherichia coli quantification in children stool samples using quantitative PCR

Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10(5) to 2 × 10(9 ) CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10(7) and 10(6) CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.

Frequency of VKORC1 (C1173T) and CYP2C9 genetic polymorphisms in Egyptians and their influence on warfarin maintenance dose: proposal for a new dosing regimen

Warfarin is one of the most widely used anticoagulants, yet interindividual differences in drug response, a narrow therapeutic range and a high risk of bleeding or stroke complicate its use. We aimed to determine the allele and genotype frequency of VKORC1 1173 C>T, CYP2C9*2 and CYP2C9*3 variant polymorphisms in the Egyptian population and to evaluate their influence on the interindividual differences in warfarin dosage. A total of 154 unrelated healthy adult patients and 46 warfarin-treated patients were included. SYBR Green-based real-time polymerase chain reaction (PCR) assay was used for studying VKORC1 (C1173T) and CYP2C9*3 polymorphisms. Mutagenically separated PCR assay was used to detect the CYP2C9*2 allele. VKORC1 genotype frequencies were 11%, 24% and 65% for CC, CT and TT, respectively. The prevalence of CYP2C9 haplotypes was 81% (*1\*1), 3.3% (*1\*2), 9.7% (*1\*3), 4.5% (*2\*2) and 0.65% (2\*3 and *3\*3). VKORC1 TT and CYP2C9*2\*2 were associated with a significantly lower warfarin dose. VKORC1 and CYP2C9 accounted for 31.7% and 15.6% of warfarin dose variability, respectively, and together with clinical factors explained 61.3% of total variability. VKORC1-TT and CYP2C9 *1/*1 are the most prevalent genotypes among Egyptians. Patients with VKORC1-TT genotype required a lower warfarin dose.

Abnormal Levels of Transcript Abundance of Developmentally Important Genes in Various Stages of Preimplantation Bovine Somatic Cell Nuclear Transfer Embryos

Based on microarray data comparing gene expression of fibroblast donor cells and bovine somatic cell nuclear transfer (SCNT) and in vivo produced (AI) blastocysts, a group of genes including several transcription factors was selected for evaluation of transcript abundance. Using SYBR green-based real-time polymerase chain reaction (Q-PCR) the levels of POU domain class 5 transcription factor (Oct4), snail homolog 2 (Snai2), annexin A1 (Anxa1), thrombospondin (Thbs), tumor-associated calcium signal transducer 1 (Tacstd1), and transcription factor AP2 gamma (Tfap2c) were evaluated in bovine fibroblasts, oocytes, embryos 30 min postfusion (SCNT), 12 h postfertilization/activation, as well as two-cell, four-cell, eight-cell, morula, and blastocyst-stage in vitro fertilized (IVF) and SCNT embryos. For every gene except Oct4, levels of transcript were indistinguishable between IVF and SCNT embryos at the blastocyst stage; however, in many cases levels of these genes during stages prior to blastocyst differed significantly. Altered levels of gene transcripts early in development likely have developmental consequences downstream. These results indicate that experiments evaluating gene expression differences between control and SCNT blastocysts may underestimate the degree of difference between clones and controls, and further offer insights into the dynamics of transcript regulation following SCNT.

Abundance of denitrifying genes coding for nitrate ( narG), nitrite ( nirS), and nitrous oxide ( nosZ) reductases in estuarine versus wastewater effluent-fed constructed wetlands

Constructed and estuarine wetlands, influenced by wastewater treatment plants, were investigated, with respect to microbial activity in terms of functional genes. The density and abundance of three denitrifying genes coding for nitrate ( narG), nitrite ( nirS), and nitrous oxide ( nosZ) reductases, in sediment soil samples from wastewater effluent-fed and estuarine wetlands, were quantified using the SYBR green-based real-time polymerase chain reaction (PCR). To assess seasonal effects (i.e., winter (average temperature ∼2 °C) versus spring (average temperature ∼20 °C)), the densities of denitrifying genes, with respect to the abundance of functional genes, for the two different wetlands were determined. The three functional genes for all the sampling sites ranged from 1.0 × 10 6 to 1.0 × 10 9 copies/g of soil. Without considering seasonal variation, the nitrite-reducing functional genes were dominant over the other two genes in the effluent-fed wetland samples. However, nitrate and nitrite-reducing functional genes were dominant in relatively cold and warm seasons, respectively, in the estuarine wetland samples. Even though robust patterns and conclusions could not be obtained from the limited investigations, patterns with certain trends and needs for potential future research directions were obtained.

Development of a real-time PCR for the differentiation of the G1 and G2/G3 genotypes of Echinococcus granulosus

The present study was aimed at developing a SYBR Green-based real-time polymerase chain reaction (PCR) assay for a rapid differentiation of the genotype G1 from the cluster of genotypes G2/G3 of Echinococcus granulosus, using as marker the 12S mtDNA gene. Eleven hydatid cysts from water buffaloes and 19 from cattle were used. Fourteen samples (identified as G1 using sequencing) showed a mean melting temperature (T (m)) of 76.4 degrees C and 16 samples (identified as G2/G3 using sequencing) showed a mean T (m) of 77.0 degrees C. The detected mean difference of the T (m) of 0.6 degrees C between G1 and G2/G3 genotypes might allow a fast and simple discrimination of these genotypes. In conclusion, the real-time PCR developed in the present study provides a powerful tool for molecular studies on E. granulosus with possibilities for extension to other genotypes using different molecular targets.

Detection of Five Avian Eimeria Species by Species-Specific Real-Time Polymerase Chain Reaction Assay

To detect five different avian Eimeria species, we applied the SYBR Green-based real-time polymerase chain reaction (PCR) assay for the diagnosis of field-isolated parasites by using their individual species-specific primer sets. The primer sets were originally designed for Eimeria acervulina, E brunetti, E necatrix, and E tenella based on the sequence of the internal transcribed spacer 1 region of ribosomal DNA, whereas for E. maxima the primer sets were derived from sequences reported previously. The detection limit of these assays was defined at 10(2) or 10(1) oocysts depending on species. Melting curves from the real-time PCR assay showed that each species has a single peak and specific melting temperature value. Fecal samples from 32 poultry farms, which were endemic for coccidiosis, were examined using this assay. The data showed that E. brunetti was found in 21 farms, E maxima and E. necatrix in 16 farms, E. tenella in 12 farms, and E. acervulina in eight farms. This survey revealed that E brunetti was highly prevalent in Japan. This technique is not only easy and rapid but also available to detect Eimeria species specifically; therefore, it can be a valuable tool for diagnostic work for chicken coccidiosis.

RAPID DETECTION OF MYCOPLASMA CONTAMINATION IN CELL CULTURES USING SYBR GREEN-BASED REAL-TIME POLYMERASE CHAIN REACTION

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Salmonella profile in chickens determined by real-time polymerase chain reaction and bacteriology from years 2000 to 2003 in Turkey

From years 2000 to 2003, Salmonella was investigated from a total of 1785 samples comprised of chicken intestinal samples, cloacal swabs, drag swabs, litter samples and chick dust samples collected from 191 poultry breeding flocks belonging to 15 different chicken breeding stock companies in the Marmara region, Turkey by a SYBR green-based real-time polymerase chain reaction (SGBRT-PCR), by a probe-specific real-time polymerase chain reaction (PSRT-PCR) and by standardized bacteriology as described in the manual of National Poultry Improvement Plan and Auxillary Provisions, United States Department of Agriculture. Between January 2000 and July 2001, Salmonella was detected at the rates of 5.87% and 4.10% out of a total of 1242 samples by SGBRT-PCR and bacteriology, respectively. From July 2001 until December 2003, Salmonella was found at rates of 11.42% and 5.52% from a total of 543 samples by PSRT-PCR and bacteriology, respectively. The dominant Salmonella serovar was determined as Salmonella enterica subsp. enterica Serovar Enteritidis (S. Enteritidis), while serogroup C1 and C2 in 2001 and serogroup E1 in 2002 were isolated as additional serovars. As a conclusion, S. Enteritidis seems to be the major problem in poultry breeding flocks in Turkey, and both of the real-time polymerase chain reaction methods were found more sensitive than standard bacteriology for the detection of Salmonella from poultry samples.