TaqMan Real-time PCR Method Research Articles

Specific detection of duck adeno-associated virus using a TaqMan-based real-time PCR assay.

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12-0.21% and 0.62-1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA-Class I and HPA.

Selecting compatible HLA-Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA-Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA-Class I and HPA genotypes. Ten pairs of specific primers for HLA-A, HLA-B, HLA-C, HPA-1 through HPA-6w, HPA-15 and HPA-21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA-A, HLA-B and HLA-C and the TaqMan real-time PCR method in-house for the genotyping of HPA-1 through HPA-6w, HPA-15 and HPA-21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA-A, HLA-B, HLA-C and HPA-1 through HPA-6w, HPA-15 and HPA-21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA-A*11:451, HLA-A*30:01:26, HLA-B*39:201 and HLA-B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA-Class I and HPA simultaneously, which is high-throughput, rapid and accurate and could be applied to build a platelet donor bank.

Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus

Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=−3.29x+39.42, a high correlation coefficient (R2 = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay’s detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay’s superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice.

Establishment and application of TaqMan real-time PCR method for detection of Theileria annulata resistant to buparvaquone

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application.

Rapid detection of goose megrivirus using TaqMan real-time PCR technology

The aim of this study was to develop an efficient and accurate platform for the detection of the newly identified goose megrivirus (GoMV). To achieve this goal, we developed a TaqMan real-time PCR technology for the rapid detection and identification of GoMV. Our data showed that the established TaqMan real-time PCR assay had high sensitivity, with the lowest detection limit of 67.3 copies/μL. No positive signal can be observed from other goose origin viruses (including AIV, GPV, GoCV, GHPyV, and GoAstV), with strong specificity. The coefficients of variation of repeated intragroup and intergroup tests were all less than 1.5%, with excellent repeatability. Clinical sample investigation data from domestic Minbei White geese firstly provided evidence that GoMV can be transmitted both horizontally and vertically. In conclusion, since the TaqMan real-time PCR method has high sensitivity, specificity, and reproducibility, it can be a useful candidate tool for GoMV epidemiological investigation.

Dynamics of Puccinia striiformis f. sp. tritici Urediniospores and Its Relationship with Meteorological Factors in Mianyang, China.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the main diseases of wheat worldwide. Mianyang of Sichuan province in Southwest China is one of main regions for winter Pst inoculum production and spring epidemic and provides urediniospores for infecting wheat in the surrounding regions. Understanding the urediniospore dynamics is important to predict and manage stripe rust. In this study, spore trapping coupled with a TaqMan real-time quantitative PCR method was used to monitor airborne Pst urediniospores from December 2019 to December 2022 in Mianyang. Weather conditions (temperature, relative humidity, daily sunshine duration, and precipitation) were collected for the same period. These data were used to study the relationship of airborne urediniospore density with climatic conditions. The results showed that Pst urediniospores were captured all year round, and the annual peak of urediniospore densities occurred in the period from March to April in which the urediniospores accounted for the largest proportion of the annual total urediniospores. The density of urediniospores in the period of March to April was linearly related to the average sunshine duration of 20 days and average temperature of 15 days prior to the final day of a 7-day trapping period. This relationship needs to be tested in other regions where Pst can sporulate during the winter before it can be integrated with Pst infection conditions to predict rust development.

Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection.

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

Development of a 1-step TaqMan real-time PCR method for detection of the Bovine Group A Rotavirus

BackgroundThe purpose of this study was to develop a 1-step real-time quantitative fluorescence polymerase chain reaction (QF-PCR) method for detecting Bovine Group A Rotavirus (BRVA). The primers and probe were designed targeting the VP6 gene of BRVA. The standard substance was obtained through in vitro transcription. The primers, probe concentration, and annealing temperatures were optimized to determine the optimal system and conditions for the reaction. The specificity, sensitivity, and repeatability of the method were assessed and compared with a reported real-time QF-PCR method for clinical samples. ResultsThe results indicated that the detection method can achieve a sensitivity of 3.47 copies/μL and exhibit good specificity by exclusively detecting BRVA without cross-reactivity to other common pathogens in cattle and sheep. The standard curve exhibited a robust linear correlation, and the amplification efficiency was calculated to be 105%. The intra-group and inter-group coefficients of variation were less than 2%. A total of 96 clinical samples were tested and compared with the real-time QF-PCR method that was reported. The coincidence rate was 90.63% (87/96). Furthermore, the clinical samples revealed that the prevalence of BRV in cattle from Fujian Province was 85.42% (82/96). ConclusionThis study has successfully developed a 1-step real-time QF-PCR method for BRVA, which offers an efficient and sensitive technical support for the rapid diagnosis and epidemiological investigation of BRVA.

Rapid detection of pigeon Megrivirus using TaqMan real-time PCR technology

Megriviruses have been identified from fecal samples in wild pigeons in Hong Kong (China) and Hungary. In this study, the genomic sequences of pigeon Megriviruses (PiMeVs) were downloaded from GenBank and compared. Based on the genetic comparison results, a pair of primers and TaqMan probe were designed based on the conserved sequences of the 3C gene (located in the P3 gene coding region), and a TaqMan real-time PCR method (TaqMan-qPCR) was established. The standard curve of the TaqMan-qPCR had an axial intercept of 39.74 and a slope of -3.2475 with a linear correlation (R2) of 1.00 and an efficiency of 103.2%. No cross-amplification signal was found from other pigeon viruses (such as avian influenza virus, pigeon paramyxovirus type I, pigeon torque teno virus, pigeon adenovirus, and pigeon circovirus). The limit of detection concentration was 53.6 copies/μL. The intra- and interassay results were less than 1.0% based on the reproducibility test. Furthermore, field samples investigation by the established TaqMan-qPCR method showed that positive signals can be found from racing pigeon fecal samples and embryos. Thus, our data suggested that this visible TaqMan-qPCR method is sensitive, specific, and reproducible. Moreover, we first confirmed the presence of pigeon Megrivirus infection in racing pigeon embryos, indicating that the virus may be vertically transmitted. This study provides a reference basis for further understanding the epidemiology of PiMeVs.

SCN9A rs6746030 Polymorphism and Pain Perception in Combat Athletes and Non-Athletes

One of the genes associated with pain perception is SCN9A, which encodes an α-subunit of the voltage gated sodium channel, NaV1.7, a crucial player in peripheral pain sensation. It has been suggested that a common missense polymorphism within SCN9A (rs6746030; G>A; R1150W) may affect nociception in the general population, but its effects of pain perception in athletes remain unknown. Therefore, the aim of the study was to investigate the association between a polymorphism within SCN9A (rs6746030) and pain perception (pain threshold and pain tolerance) in the group of combat athletes (n = 214) and students (n = 92) who did not participate in sports at a professional level. Genotyping was carried out using TaqMan Real-Time PCR method. No significant differences were found between the SCN9A genotype distributions with respect to the pain threshold. However, the probability of having a high pain threshold was higher in the combat sports group than in the control group. The probability of having a decreased pain tolerance was higher in the carriers of the GA and AA genotype than in the homozygotes of the GG genotype. Moreover, the possibility of having a high pain threshold was higher in the combat athlete group than in the control group. The results of our study suggest that the SCN9A rs6746030 polymorphism may affect pain perception. However, the additional effect of the experimental group may suggest that pain tolerance is significantly modulated by other factors, such as the systematic exposure of the athletes' bodies to short-term high-intensity stimuli during training sessions.

Specific quantitative detection of Streptococcus suis and Actinobacillus pleuropneumoniae in co-infection and mixed biofilms.

Respiratory infections seriously affect the swine industry worldwide. Co-infections of two vital pathogenic bacteria Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae), colonizing the respiratory tract often occurs in veterinary clinical practice. Moreover, our previous research found that S. suis and A. pleuropneumoniae can form biofilm in vitro. The formation of a mixed biofilm not only causes persistent infections, but also increases the multiple drug resistance of bacteria, which brings difficulties to disease prevention and control. However, the methods for detecting S. suis and A. pleuropneumoniae in co-infection and biofilm are immature. Therefore, in this study, primers and probes were designed based on the conservative sequence of S. suis gdh gene and A. pleuropneumoniae apxIVA gene. Then, a TaqMan duplex real-time PCR method for simultaneous detection of S. suis and A. pleuropneumoniae was successfully established via optimizing the reaction system and conditions. The specificity analysis results showed that this TaqMan real-time PCR method had strong specificity and high reliability. The sensitivity test results showed that the minimum detection concentration of S. suis and A. pleuropneumoniae recombinant plasmid was 10 copies/μL, which is 100 times more sensitive than conventional PCR methods. The amplification efficiencies of S. suis and A. pleuropneumoniae were 95.9% and 104.4% with R2 value greater than 0.995, respectively. The slopes of the calibration curves of absolute cell abundance of S. suis and A. pleuropneumoniae were 1.02 and 1.09, respectively. The assays were applied to cultivated mixed biofilms and approximately 108 CFUs per biofilm were quantified when 108 CFUs planktonic bacteria of either S. suis or A. pleuropneumoniae were added to biofilms. In summary, this study developed a TaqMan real-time PCR assay for specific, accurate quantification of S. suis or A. pleuropneumoniae in mixed biofilms, which may help for the detection, prevention and control of diseases caused by a bacterial mixed infection involving S. suis and A. pleuropneumoniae.

Diagnostic performance of a Rapid Test Kit for white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV) is a highly infectious and influential pathogen that causes mass mortality. Particularly, this virus has had the most severe impact on global shrimp culture over the past 30 years. There are currently no vaccines or treatments for WSSV, and therefore accurate diagnosis strategies are critical for the management of shrimp farms. In this study, a commercial WSSV lateral flow immunoassay (WSSV RP Rapid Test Kit, Innocreate Bioscience Co., Ltd., Taiwan) can be used to assess the level of WSSV infection in shrimp farms was tested. Sensitivity analyses indicated that 0.4 ng of a recombinant target protein could be reliably detected after 15 min. Additionally, this kit did not return false-positive reactions when testing samples infected with PirABVP-producing strain of Vibrio parahaemolyticus (the causative agent of acute hepatopancreatic necrosis disease, AHPND), haematopoietic necrosis virus (IHHNV), and Enterocytozoon hepatopenaei (EHP). Furthermore, the accuracy and sensitivity of our proposed procedure were assessed using the OIE TaqMan real-time PCR method. Upon analyzing 231 samples, our WSSV immunoassay achieved a sensitivity and specificity of 99.21% and 100%, respectively. Collectively, our findings demonstrated the convenience, stability, sensitivity, and specificity of the WSSV RP Rapid Test Kit, and therefore this kit can be widely used in aquaculture farms to assist disease management.

CER 1 gene polymorphism in postmenopausal Roma and non-Roma Slovak women in connection with osteoporosis.

The aim of our study was to investigate the relationship between the rs74434454 polymorphism of the CER1 gene and selected biochemical, densitometric and anthropometric markers in Slovak postmenopausal women of two ethnic groups: Roma and non-Roma. The scientific study included 303 postmenopausal women of the non-Roma and Roma populations who were divided into two groups based on densitometric measurements: control group (CG) and osteoporotic group (OG). Genomic DNA was isolated from peripheral blood using a commercial NucleoSpin® Blood kit following a standard protocol. The TaqMan Real-Time PCR method was used for genotyping. Biochemical markers were measured with Cobas e411 and Cobas Integra400 plus analysers. In the control group of postmenopausal Roma women, the occurrence of the risk genotype GG was not observed. In the group of Roma women with osteopenia and osteoporosis, the GG genotype occurred at a frequency of 3.03%. In the group of non-Roma women (between CG and OG) statistically significant differences were found in all monitored biochemical markers except CTx-I (p<0.66). In contrast, in the group of Roma women, statistical significance was only found in the osteoresorption marker CTx-I (p<0.007). In the population of Roma women, we did not find a statistically significant difference between the AA, AG and GG genotypes in any of the monitored markers. The results provide the first and unique insight on the distribution of genotypes and alleles of the rs74434454 CER1 gene polymorphism and its relationship to markers of bone metabolism in two ethnically distinct groups.

Investigating the association between rs6983267 polymorphism and susceptibility to gastrointestinal cancers in Iranian population.

Genome-wide association studies have revealed that some single nucleotide polymorphisms at 8q24, such as rs6983267, might be effective in susceptibility to various cancers in different populations. Therefore, rs6983267 might be useful as a marker for multiple cancers. In this study, we considered a population, including 478 gastrointestinal cancer cases from the Iranian population, to investigate the association between rs6983267 and susceptibility to gastrointestinal cancers. The samples were genotyped using the TaqMan real-time PCR method while 10% of them were also confirmed by sequencing. Higher frequency of G allele was associated with higher grades of tumors in esophageal cancer and the tumors located in the lower portion of the esophagus (OR 3.56; 95% CI 1.13-11.24; P = 0.03) and cardia (OR 5.24; 95% CI 1.26-21.83; P = 0.02), which both locations are involved in esophageal adenocarcinomas with poor prognosis. The results indicated that in the male subgroup, the rs6983267 GG genotype significantly enhanced the gastric cancer susceptibility (OR 4.76; 95% CI 1.57-14.45; P = 0.01). GG genotype alsoincreased the risk of intestinal-type gastric cancer, located in non-cardia (OR 4.62; 95% CI 1.25-17.04; P = 0.02). Moreover, gastric cancer cases and controls with a family history of gastrointestinal tumors were mostly genotyped with the G allele (OR 3.61; 95% CI = 1.09-12.01; P = 0.04). There were no remarkable associations between rs6983267 and susceptibility to esophageal and colon cancers in the Iranian population. However, different genotypes of rs6983267 had significant correlations with tumor grade, cancer type, and family history of gastrointestinal cancers. Further investigations in a larger population and other ethnicities are required to confirm these results.

The role of the IRS1 gene (rs2943640) in the comorbid course of type 2 diabetes mellitus, obesity and arterial hypertension

Type 2 diabetes mellitus (T2DM) affects more than 7 million people, resulting in 2.8 million hospitalizations and more than 300,000 deaths reported annually. Current scientific data indicate that among the world's population, arterial hypertension (AH) and type 2 diabetes mellitus (T2DM) after obesity are among the leading factors of cardiovascular risk.&#x0D; Aim of research: was to establish the prevalence of the IRS1 gene (rs2943640) polymorphism in patients with T2DM in combination with obesity and arterial hypertension/&#x0D; Material and research methods. The study involved 33 type-2 diabetic patients hospitalized to the Endocrinology Department of Ternopil University Hospital (Ternopil, Ukraine) in 2019-2020 and 10 healthy individuals.&#x0D; Inclusion criteria: clinical, laboratory and instrumental signs of T2DM, AH and obesity. Exclusion criteria from the study: signs of clinically significant neurological, mental, renal, hepatic, immune, gastrointestinal, urogenital disorder; injuries of the musculoskeletal system, skin, sense organs, endocrine system (except T2DM); or uncontrolled hematologic diseases; acute pancreatitis, unstable or life-threatening heart disease; patients with malignant neoplasms who have not been in complete remission for at least 5 years, medication (drug) dependence, and alcohol dependence.&#x0D; T2DM diagnoses were confirmed according to the 2019 Recommendations of the American Diabetes Association (ADA). The diagnosis of arterial hypertension (Stage I) was made according to 2018 ESC/ESH Guidelines for the management of arterial hypertension. Systolic (140-159 mmHg) and/or diastolic (90-99 mmHg) blood pressure were considered as the presence of Stage I AH. Left ventricular hypertrophy was confirmed by an electrocardiogram.&#x0D; Genomic DNA was extracted from peripheral blood leukocytes using a commercially available DNA isolation kit (QIAamp Blood DNA Mini Kit, QIAGEN, Germany). The IRS-1 gene rs2943640 C&gt;A polymorphism was genotyped using the TaqMan real-time PCR method (Applied Biosystems, Foster City, CA, USA).&#x0D; Statistical analysis of the data was performed using the software STATISTICA 7.0.&#x0D; Results. The frequency distribution of the IRS1 gene (rs2943640) polymorphisms and the assessment of compliance with the Hardy – Weinberg equilibrium were performed in the experimental and control groups. It was found that the frequencies of the genotype responsible for C/A polymorphism of the IRS1 gene at T2DM, T2DM with obesity and in the combined course of T2DM with obesity and arterial hypertension did not deviate from the Hardy – Weinberg equilibrium (p&gt; 0.05), while in the control group the selected sample did not correspond to the general population.&#x0D; The corresponding frequencies for the genotypes of the IRS1 gene were as follows: 66.7% for C/A and 33.3% for A/A in the experimental group 1; 42.9% for C/C, 57.1% for C/A in group 2; 47.1% for C/C, 29.4% for C/A and 23.5% for A/A in group 3 and 100.0% for C/A in the control group.&#x0D; Analysis of allele frequencies for the IRS1 gene in patients with T2DM and comorbidity showed that in patients with T2DM the A allele prevailed (2.0 times), while in patients with T2DM + obesity and T2DM + obesity + arterial hypertension – the C allele. It should be noted that the C allele and the A allele were equally present in the control group.&#x0D; Analysis of the odds ratio for IRS1 gene genotypes (rs2943640) in patients with T2DM, T2DM and obesity showed no statistically significant relationship between factor (presence of C or A alleles) and disease onset (p&gt; 0.05). At the same time, the significant influence of the C/A genotype of the IRS1 gene on the development of T2DM combined with obesity and arterial hypertension (p &lt;0.05) was established. This is confirmed by a significant difference in the dominant model of inheritance of the IRS1 gene only in the group with the combination of T2DM with obesity and arterial hypertension compared with the control group (reliability coefficient for the chi-square p &lt;0.001).&#x0D; Conclusion. The presence of the C allele of the IRS1 gene (rs2943640) in both homozygous and heterozygous states may increase the risk of comorbid course of T2DM, obesity and arterial hypertension.

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

Background and Aim:Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and Methods:One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.Results:TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.Conclusion:A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Relationship between MEG3 gene polymorphism and risk of gastric cancer in Chinese population with high incidence of gastric cancer.

Objective: Gastric cancer is the most common gastrointestinal malignancy in China and results from a combination of genetic and environmental factors. The present study was conducted to investigate the relationship between long noncoding RNA (lncRNA) materally expressed gene 3 (MEG3) single nucleotide polymorphisms (SNPs) and the risk of gastric cancer and to construct a genetic-environmental risk assessment model. Methods: A case–control study was conducted to include 474 patients with gastric cancer diagnosed by clinical and pathological examination and 543 healthy physical examination subjects. Blood samples, general demographic data and behavioral lifestyle of the subjects were collected. The TaqMan real-time PCR method was used for testing the genotypes of MEG3 rs7158663 and rs10132552. Results: The A allele at the rs7158663 loci of MEG3 was found to be risk factor for gastric cancer (odds ratio (OR) = 1.41, 95% confidence interval (95% CI) = 1.14–1.74, P=0.002). Yet, no significant association between rs10132552 polymorphisms and gastric cancer was observed. Drinking, tea drinking and preserved food eating were risk factors for gastric cancer (P<0.05). A genetic–environmental risk assessment model was established by using the logistic regression model to include MEG3 rs7158663, drinking, tea drinking, and preserved food eating. With the increase in risk score (RS), the risk of gastric cancer increased substantially (P<0.05). And the area under the receiver operating characteristic (ROC) curve was 0.745, which indicates a high diagnostic value. Conclusions: MEG3 rs7158663 might be associated with the risk of gastric cancer; the diagnostic ability of genetic–environmental risk assessment model for gastric cancer is better.

Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

BackgroundAfrican swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.ResultsIn this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.ConclusionWe successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

Establishment and Application of a Dual TaqMan Real-Time PCR Method for Proteus Mirabilis and Proteus Vulgaris

Proteus species are common opportunistic bacteria and foodborne pathogens. The proper detection of Proteus can effectively reduce the occurrence of food-borne public health events. Proteus mirabilis and Proteus vulgaris are the two most important pathogens in the Proteus genus. In this study, a dual TaqMan Real-Time PCR method was established to simultaneously detect and distinguish P. mirabilis and P. vulgaris in samples. The method exhibited good specificity, stability, and sensitivity. Specifically, the minimum detection concentrations of P. mirabilis and P. vulgaris in pure bacterial cultures were 6.08 × 102 colony forming units (CFU)/ml and 4.46 × 102 CFU/ml, respectively. Additionally, the minimum detectable number of P. mirabilis and P. vulgaris in meat and milk was 103 CFU/g. In addition, the method can be used to distinguish between strains of P. mirabilis and P. vulgaris within two hours. Overall, it is a sensitive, easy-to-use, and practical test for the identification and classification of Proteus in food.

Reverse expression pattern of sirtuin-1 and histone deacetylase-9 in coronary artery disease

Background SIRT1 and HDAC 9 genes are related to inflammation and may contribute to the pathogenesis of coronary artery disease (CAD). We aimed to evaluate the expression level, methylation profile and polymorphisms of these genes in CAD patients. Methods In this study, 50 CAD patients and 50 healthy individuals were recruited. The expression level change was evaluated using the TaqMan Real-Time PCR method. The methylation of genes promoter and genotyping of polymorphisms were evaluated by the HRM. Results The expression level of SIRT1 was reduced while the HDAC9 expression level showed a significant elevation (p < .001). The SIRT1 gene promoter was hypomethylated and the HDAC9 gene promoter was hypermethylated in CAD patients. Also, CG + GG genotype in SIRT1 and both genotypes in the HDAC9 gene were associated with expression change. Conclusions SIRT1 and HDAC9 genes, expression changes can be suggested as a potential biomarker for CAD detection.