Duplex Real-time Polymerase Chain Reaction Research Articles

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/μL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

Development of a rapid quantitative method to differentiate MS1 vaccine strain from wild-type Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an economically important pathogen in the poultry industry. Vaccination is an effective method to prevent and control MS infections. Currently two live attenuated MS vaccines are commercially available, the temperature-sensitive MS-H vaccine strain and the NAD-independent MS1 vaccine strain. Differentiation of vaccine strains from wild-type (WT) strains is crucial for monitoring MS infection, especially after vaccination. In this study, we developed a Taqman duplex real-time polymerase chain reaction (PCR) method to identify MS1 vaccine strains from WT strains. The method was specific and did not cross-react with other avian pathogens. The sensitivity assay indicated that no inhibition occurred between probes or between mixed and pure templates in duplex real-time PCR. Compared with the melt-based mismatch amplification mutation assay (MAMA), our method was more sensitive and rapid. In conclusion, the Taqman duplex real-time PCR method is a useful method for the diagnosis and differentiation of WT-MS and MS1 vaccine strains in a single reaction.

Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and Anguillid herpesvirus-1

Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1) are virulent pathogens in eel aquaculture farms in the Republic of Korea and coexist in the same host. To date, no diagnostic method has been developed that detects them simultaneously. We developed a duplex real-time polymerase chain reaction (duplex qPCR) assay to detect both JEECV and AngHV-1 simultaneously in a single reaction. Two sets of specific primers and TaqMan probes were designed to target the large T-antigen gene of JEECV and the DNA polymerase catalytic subunit gene of AngHV-1. The assay was optimized by changing the primer and probe concentrations and the annealing temperature of the reaction. The duplex qPCR could specifically amplify the specific genes of the two viruses with high efficiency and coefficient of determination. The assay could detect both viruses at a limit of detection of 8 fg/μL. The diagnostic performance of the developed assay was determined by analyzing 82 clinical samples (72 infected and 10 healthy) and comparing the results with those of conventional PCR and SYBR Green-based qPCR; the results correlated well with each other. The duplex qPCR showed 100% and 98.6% diagnostic sensitivity for JEECV and AngHV-1, respectively. The diagnostic specificity of this assay was validated using two potential viral strains, red sea bream iridovirus and Ostreid herpesvirus-1 microvariant, reported in the aquaculture industry. In contrast, the methods already developed including conventional PCR and qPCR cannot detect both JEECV and AngHV-1 at the same time as they should perform in separate reactions. Thus, the duplex qPCR developed herein is a sensitive and specific diagnostic tool for the simultaneous detection of JEECV and AngHV-1 and has subsequent applications in the health management of eels.

Quantification of Pork, Chicken, Beef, and Sheep Contents in Meat Products Using Duplex Real-Time PCR.

Accurate methods for meat speciation and quantification are essential for ensuring the supply of safe and wholesome meat and composite products with animal origins to negate the potential associated hazards, aid classification of consignments at the import control system, and thwart food fraud committed for financial gain. To better enhance meat safety control and combat food fraud, this study developed two duplex real-time polymerase chain reaction (real-time PCR) systems specifically designed for chicken, pork, sheep, and beef, using single-copy, chromosomally encoded, species-specific gene sequences to accurately measure the content of each meat type in meat products. DNA extracted from the raw and boiled reference materials prepared in varying proportions (ranging from 1% to 75%) were used in the development of the duplex assay to derive calibration factors to determine the meat content in different meat products. The method was further validated using proficiency test samples and market monitoring samples. Our findings showed that this method exhibits high specificity and sensitivity, with a significant accuracy range of 0.14% to 24.07% in quantifying the four meat types in both raw and processed meat products. Validation results further confirmed the effectiveness of our method in accurately quantifying meat content. Thus, we have demonstrated the duplex qPCR assays as promising approaches for implementation in routine analysis to strengthen meat safety control systems and combat meat fraud, thereby safeguarding consumer health and trust in the meat industry.

Detecting the Minimum Limit on Wheat Stripe Rust in the Latent Period Using Proximal Remote Sensing Coupled with Duplex Real-Time PCR and Machine Learning.

Wheat stripe rust (WSR) is an airborne disease that causes severe damage to wheat. The rapid and early detection of WSR is essential for the prevention and control of this disease. The minimum detection limit (MDL) is one of the most important characteristics of quantitative methods that can be used to determine the scope and applicability of a measurement technique. Three wheat cultivars were inoculated with Puccinia striiformis f.sp. tritici (Pst), and a spectrometer was used to collect the canopy hyperspectral data, and the Pst content was obtained via a duplex real-time polymerase chain reaction (PCR) during the latent period, respectively. The disease index (DI) and molecular disease index (MDI) were calculated. The regression tree algorithm was used to determine the MDL of the Pst based on hyperspectral feature parameters. The logistic, IBK, and random committee algorithms were used to construct the classification model based on the MDL. The results showed that when the MDL was 0.7, IBK had the best recognition accuracy. The optimal model, which used the spectral feature R_2nd.dv ((the second derivative of the original hyperspectral value)) and the modeling ratio 2:1, had an accuracy of 91.67% on the testing set and 90.67% on the 10-fold cross-validation. Thus, during the latent period, the MDL of Pst was determined using hyperspectral technology as 0.7.

Differential detection of sheep and goat meat using duplex real-time PCR and high-resolution melt analysis

Authenticity in meat and meat products is of paramount importance in preventing adulteration and ensuring food safety. Since morphologically indistinct samples like sheep and goat meat requires accurate and sensitive differentiation, we developed and validated a duplex real-time polymerase chain reaction (qPCR) assay with high-resolution melt analysis (HRMA) for simultaneous identification of sheep and goat meat. Species-specific amplicons with melting temperature of 78 °C for goat and 80 °C for sheep (with associated standard errors) was identified. The assay was optimized for basic PCR parameters and the HRMA detection was standardized based on melt curve shape, color and inflexion points. The absolute limit of detection (LODabs) and limit of quantification (LOQ) of the assay were found to be 0.39 ng and 0.78 ng of target DNA respectively. A sensitivity of 0.1% was obtained for both sheep and goat meat. The relative limit of detection (LODrel) was also found to be 5%. The linearity and efficiency were evaluated in both simplex and duplex assays. The assay applicability was evaluated using proficiency test samples and commercially available raw and processed meat samples. The obtained results showed that this closed-tube qPCR-HRMA method can be a rapid and sensitive detection technique for meat speciation.

Development and application ofalow-priced duplex quantitative PCR assay based onSYBR GreenI for the simultaneous detection ofporcine deltacoronavirus and porcine sapelovirus.

Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea inpigs and bring great economic loss tothe pig industry. Inthis research, aduplex real-time quantitative polymerase chain reaction (qPCR) assay based onSYBR Green I was developed tosimultaneously detect PDCoV and PSV. Nospecific melting peaks were found inother porcine diarrhoea-associatedviruses, indicating that the method developed inthis study had good specificity. The detection limits ofPDCoV and PSV were 1.0×101copiesμl-1 and 1.0×102 copiesμl-1, respectively. The duplex real-time qPCR assay tested two hundred and three (203) intestinal and faecal samples collected from diarrhoeal and asymptomatic pigs. The positive rates ofPDCoV and PSV were 20.2% and 23.2%, respectively. The co-infection rate ofPDCoV and PSV was 13.8%. Toevaluate the accuracy ofthedeveloped method, conventional PCR and singular TaqMan real-time qPCR assays for PDCoV/PSV were also used todetect the samples. The results showed that the duplex real-time qPCR assay was consistent with thesingular assays, but its sensitivity was higher than conventional PCR methods. This duplex real-time qPCR assay provides arapid, sensitive and reliable method inaclinic tosimultaneously detect PDCoV and PSV.

Molecular Detection of Porcine Parainfluenza Viruses 1 and 5 Using a Newly Developed Duplex Real-Time RT-PCR in South Korea.

Two species of porcine parainfluenza viruses (PPIV), PPIV1 and PPIV5, are globally distributed in pig herds and associated with porcine respiratory diseases, and a diagnostic tool for the simultaneous detection of the two viruses is required. In this study, a TaqMan probe-based duplex real-time reverse transcription polymerase chain reaction (dqRT-PCR) assay was first developed for the differential detection of PPIV1 and PPIV5 nucleocapsid protein (NP) genes in porcine clinical samples. The dqRT-PCR assay was highly sensitive, its limit of detection was approximately 10 RNA copies/reaction, it specifically amplified the targeted NP genes of PPIV1 and PPIV5 without cross-reacting with other porcine pathogens, and their clinical detection rates were 15.2% and 0.7%, respectively. The results from 441 clinical samples taken from 278 Korean domestic pig farms showed that the prevalence of PPIV1 and PPIV5 was 11.2% and 1.1%, respectively, and co-infection of both viruses was confirmed in a farm, suggesting that PPIV1 and PPIV5 are co-circulating in current Korean pig herds. Phylogenetic analysis based on the partial NP genes suggested that genetically diverse PPIV1 strains are circulating in Korean pig herds. The developed dqRT-PCR assay was found to be an accurate, reliable, and quantitative detection tool for PPIV1 and PPIV5 RNA in clinical pig samples and will be useful for etiological and epidemiological studies and the control of viral infections in the field.

Machine Learning-Assisted Real-Time Polymerase Chain Reaction and High-Resolution Melt Analysis for SARS-CoV-2 Variant Identification.

Since the declaration of COVID-19 as a pandemic in early 2020, multiple variants of the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) have been detected. The emergence of multiple variants has raised concerns due to their impact on public health. Therefore, it is crucial to distinguish between different viral variants. Here, we developed a machine learning web-based application for SARS-CoV-2 variant identification via duplex real-time polymerase chain reaction (PCR) coupled with high-resolution melt (qPCR-HRM) analysis. As a proof-of-concept, we investigated the platform's ability to identify the Alpha, Delta, and wild-type strains using two sets of primers. The duplex qPCR-HRM could identify the two variants reliably in as low as 100 copies/μL. Finally, the platform was validated with 167 nasopharyngeal swab samples, which gave a sensitivity of 95.2%. This work demonstrates the potential for use as automated, cost-effective, and large-scale viral variant surveillance.

An improved duplex real-time PCR method for the systematic detection of commercially relevant crustaceans in food

Crustaceans are known food allergens that can cause severe reactions for allergic patients. Hitherto, methods to detect commercial relevant crustacean species often consist of many PCR systems. In this study, we developed a highly specific duplex PCR (polymerase chain reaction) method for the detection of most commercially relevant crustaceans in food. The nuclear 18S rRNA and the mitochondrial 16S rRNA genes were selected for the detection of the suborders Pleocyemata and Dendrobranchiata, respectively. Both suborders belong to the order Decapoda, which comprises about 96% of all crustaceans consumed as food. We cross-tested over 100 different plant and animal species, including 25 species of crustaceans and eight species of farmed insects. Eight different food products were tested, to ensure practical applicability. The limit of detection (LOD12) is 1 pg of crustacean DNA or 10 ppm (mg/kg) of crustacean sample in food matrix. Finally, a robustness test confirmed the practical use of the protocol.

Development and Application of a Duplex Droplet Digital Polymerase Chain Reaction Assay for Detection and Differentiation of EP402R-Deleted and Wild-Type African Swine Fever Virus.

African swine fever (ASF) is a highly fatal porcine disease caused by the African swine fever virus (ASFV), and resulting in huge economic losses across the globe. ASF has been raging in China for 3 years, and recently EP402R-deleted ASFV strains emerged, showing sub-acute or chronic symptoms in pigs and providing novel difficulties to monitor and control the disease as EP402R-deleted strains possess no hemadsorption (HAD) ability. In addition, the gene deletion virus with low viral load is prone to results retest or false negative due to the high cycle threshold (Ct) value under the current real-time polymerase chain reaction (PCR) detection method. Thus, a new method is needed to detect and distinguish wild strains and gene-deleted viruses. In this study, a duplex droplet digital polymerase chain reaction (ddPCR) assay based on the ASFV B646L and EP402R genes was established and showed good linearity (R2 > 0.99). The limit of detection for duplex ddPCR was 52 copies per reaction and 8.6 copies per reaction for B646L and EP402R, respectively. No cross-reaction with other porcine viruses [classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV2)] was identified by this assay. In addition, 44 ASFV-suspicious clinical samples as well as EP402R-deleted ASFV were tested in parallel by duplex real-time PCR and ddPCR, indicative of a higher sensitivity which belonged to the duplex ddPCR assay. In summary, this is the first time that duplex ddPCR assay has been successfully developed to provide an efficient method to detect and differentiate ASFV wild-type and gene-deleted strains.

Development of a Duplex TaqMan Real-Time Polymerase Chain Reaction for Accurate Identification and Quantification of Salmonella Enteritidis from Laboratory Samples and Contaminated Chicken Eggs.

Salmonella enteritidis is a major causative agent of foodborne illnesses worldwide. As the traditional serotyping and quantification methods are labor-intensive, time-consuming, and expensive, faster and more convenient molecular diagnostic methods are needed. In this study, we developed and validated a rapid duplex TaqMan real-time polymerase chain reaction (PCR) for the accurate identification and quantification of S. enteritidis. The primers and TaqMan probes were designed based on the S. enteritidis-specific gene lygD and the Salmonella genus-specific gene invA. The melt curve and gel electrophoresis analysis showed that the designed primers had potent specificity for the amplification of lygD and invA. The duplex real-time PCR specifically identified S. enteritidis from a panel of 40 Salmonella strains that represented 29 serovars and 12 non-Salmonella organisms. The duplex real-time PCR assay detected four copies of S. enteritidis DNA per reaction. The intra- and inter- assays indicated a high degree of reproducibility. The real-time PCR could accurately detect and quantify S. enteritidis in chicken organs after Salmonella infection. Furthermore, the assay identified 100% of the S. enteritidis and Salmonella genus isolates from chicken egg samples with superior sensitivity after 6 h of pre-enrichment compared to the traditional culture method. Additionally, the most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT) method was developed for the population determination of S. enteritidis and compared with various enumeration methods. Thus, we have established and validated a new duplex real-time PCR assay and MPN-qPCR-SIT method for the accurate detection and quantification of S. enteritidis, which could contribute to meeting the need for fast detection and identification in prevention and control measures for food safety.

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma.

Background:The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype.Methods:The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma.Results:Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed.Conclusion:The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus.

Simultaneous detection of feline parvovirus and feline bocavirus using SYBR Green I-based duplex real-time polymerase chain reaction.

Since both feline parvovirus (FPV) and feline bocavirus (FBoV) can cause diarrhea in cats, it is difficult to distinguish them clinically. This study aimed to develop a SYBR Green I-based duplex real-time polymerase chain reaction (PCR) assay for distinguishing FPV and FBoV-1 on the basis of the melting temperature of the PCR product. A total of 132 fecal samples from different domestic and feral cats were collected, and the results of SYBR Green I-based duplex real-time PCR assay were compared with those of the traditional PCR assay for a comprehensive evaluation. The melting temperatures were found to be 86 °C and 77.5 °C for FBoV-1 and FPV, respectively, and no specific melting peaks for other non-targeted feline viruses were observed. The data obtained from this assay had a good linear relationship; the detection limits of FPV and FBoV-1 were 2.907 × 101 copies/μL and 3.836 × 101 copies/μL, respectively. In addition, the experiment exhibited high reproducibility. The positive detection rates of the SYBR Green I-based duplex real-time PCR assay for FPV and FBoV-1 were 16.67% (22/132) and 6.82% (9/132), respectively, and the positive detection rate for co-infection with FPV and FBoV-1 was 3.03% (4/132). This result was much more sensitive than that of the traditional PCR method. Thus, the developed SYBR Green I-based assay is a sensitive, rapid, specific, and reliable method for the clinical diagnosis of FPV and FBoV-1 and can provide technical support for the simultaneous detection of co-infection with these viruses in the future.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13205-021-02947-w.

Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis

BackgroundCanine babesiosis and anaplasmosis, caused by Babesia canis and Anaplasma phagocytophilum, respectively, are significant tick-borne diseases in Baltic countries. Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of B. canis and A. phagocytophilum in canine clinical samples.MethodsSequence-based polymorphism analysis of genes encoding B. canis-specific merozoite surface protein Bc28.1 (Bc28.1) and A. phagocytophilum malate dehydrogenase (mdh) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay.ResultsThe presence of three B. canis Bc28.1 gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of A. phagocytophilum mdh genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for B. canis and A. phagocytophilum. The analytical sensitivity of this assay was ten gene copies/ reaction for both pathogens.ConclusionsA novel duplex RT-PCR molecular method was developed for the detection of B. canis and A. phagocytophilum in canine clinical samples. Sequence variability of Bc28.1 and mdh genes indicated the genetic variability of B. canis and A. phagocytophilum isolates occurring in Latvian domestic dogs.Graphical

Applicability of plasmid calibrant pMON87712 for quantitative detection of the transgenic soybean MON87712.

The transgenic glyphosate-tolerant soybean MON87712 event was developed by the agrochemical and agricultural biotechnology company Monsanto (USA) and commercialized in 2013. Due to the absence of matrix-based and genomic DNA-positive reference material for MON87712, it is very difficult to detect and monitor this event. In this study, we developed a recombinant 760-bp linearized plasmid, including 150 bp of the soybean endogenous lectin gene and 610 bp of the exogenous BBX32 gene plus its 3' flanking sequence of MON87712 by In-Fusion cloning technology. In addition, a duplex real-time polymerase chain reaction for the detection of MON87712 and the soybean endogenous lectin gene was established. By using this method, we achieved specific and quantitative detection of MON87712 in 45 other kinds of crops, with a detection limit of 10 copies/μl. This method provides a new technical means for the accurate detection of transgenic soybean MON87712, as well as technical support for the supervision of agricultural transgenic organisms.

Severity of parechovirus infections in infants under 3 months of age and comparison with enterovirus infections: A French retrospective study

While enteroviruses (EV) are a well-recognized cause of aseptic meningitis in children, human parechoviruses (HPeV), especially genotype 3, have been increasingly reported as a frequent cause of sepsis-like illness and meningitis among young infants. The aim of this study was to describe the epidemiological, clinical, and laboratory characteristics of HPeV infections in infants and to compare them with those of well-known EV infections. This monocentric retrospective study was carried out at the pediatric unit of Nantes University Hospital from January 2015 to August 2018. All patients under 18 years of age with diagnosis codes referring to fever, for whom viral infection was suspected and cerebrospinal fluid (CSF) specimens were collected, were included. All CSF specimens were screened by duplex real-time polymerase chain reaction (PCR) assay that allows for the simultaneous detection of EV and HPeV in clinical samples. During the study period, 1373 CSF specimens from patients under 18 were included. A total of 312 CSF samples were positive for HPeV (n=34) or EV (n=278). Among the 34 HPeV-positive patients, 97% (33/34) were under 3 months of age, whereas the rate was 54% (149/278) for EV-positive patients (P<0.001); thus, patients under 3 months of age were defined as the study population for the rest of this work. A review of the medical records was carried out for the positive cases. In this population, the HPeV detection rate was 5.6% versus 25.3% (P<0.001) for EV. All but one of the HPeV samples available for genotyping were HPeV-3. No seasonality was observed for HPeV infections. Length of hospital stay tended to be longer for children infected with HPeV compared with those infected by EV (3 days vs. 2 days, P=0.05). Clinicians reported more severe illness presentations among HPeV-infected infants, with more frequent administration of fluid bolus (P<0.02). Regarding laboratory characteristics, a significant lack of cellular reaction in the CSF (P=0.004) as well as lower C-reactive protein (CRP) levels (P=0.006) and neutrophil counts (P<0.001) were noted for HPeV infections compared with EV infections. Our results confirm the early onset of HPeV infections (more than 95% of patients aged under 3 months). The clinical presentation and laboratory characteristics of the two infections was similar. However, some higher clinical severity criteria and a lack of CSF pleocytosis were regularly observed in patients with HPeV infections. Considering the significant proportion (5.6%; 95% CI, 3.7–7.5) of all CSF samples in our series, HPeV detection should be systematically included in the microbiological diagnosis of febrile children under 3 months of age.

PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

BackgroundThe prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs.ResultFirst, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays.ConclusionIn this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

Simultaneous detection of duck circovirus and novel goose parvovirus via SYBR green I-based duplex real-time polymerase chain reaction analysis

Beak atrophy and dwarfism syndrome (BADS) is commonly caused by co-infection with duck circovirus (DuCV) and novel goose parvovirus (NGPV). Therefore, concurrent detection of both viruses is important for monitoring and limiting BADS, although such a diagnostic test has not been reported. In this study, we developed a duplex, SYBR Green I-based real-time polymerase chain reaction (PCR) assay to enable the simultaneous detection of DuCV and NGPV. The assay readily distinguished between the two viruses, based on their different melting temperatures (Tm), where the Tm for DuCV was 80 °C and that for NGPV was 84.5 °C. Other non-target duck viruses that were tested did not show melting peaks. The detection limit of the duplex assay was 101 copies/μL for both viruses. This method exhibited high repeatability and reproducibility, and both the inter-assay and intra-assay variation coefficients were <1.6%. Thirty-one fecal samples were collected for clinical testing using real-time PCR analysis, and the results were confirmed using sequencing. The rate of co-infection was 6.5%, which was consistent with the sequencing results. This duplex real-time PCR assay offers advantages over other tests, such as rapid, sensitive, specific, and reliable detection of both viruses in a single sample, which enables the quantitative detection of DuCV and NGPV in clinical samples. Using this test may be instrumental in reducing the incidence of BADS and the associated economic losses in the duck and goose industries.

Duplex-real time polymerase chain reaction assay for simultaneous analysis of pork and chicken in sausage products

The adulteration of meat-based food is common due to the price difference among raw meat materials. One of the favorite foods commonly consumed by Indonesian and Malaysian societies is chicken sausage, which can be substituted by pork to get economical profits. The objective of this study was to develop a duplex real-time PCR assay using the EvaGreen fluorescence dye for the identification of chicken and pork in sausage products. The method involved the application of chicken (Gallus gallus) and pork (Sus scrofa) specific primers which amplify the small fragments (pork 176 bp and chicken 183 bp) of the mitochondrial D-loop 22 and mt-12s rRNA genes, respectively. DNA was isolated from raw meat materials and reference sausage made from the mixtures of chicken and pork to optimize the assay. The primers used for pork were forward 5’- TCG TAT GCA AAC CAA AAC GCC -3’ and reverse: 5’- ATG CAT GGG GAC TAG CAG TTA -3’, while primers used for chicken were forward: 5’ TGA GAA CTA CGA GCA CAA AC 3’ and reverse: 5’ ACA TTG TGG GAT CTT CTA GGT 3’. Gene products of chicken and pork produced two distinct melting peaks simultaneously at 76.5 and 84.5oC, respectively. The detection limit of duplex-real time PCR analysis of the reference sausage samples was 0.5% for pork and chicken meat in sausage products. The coefficient of variation (CV) of threshold cycles (Ct) for amplification was 6.25%, lower than that required by the Codex Alimentarius Commission. Duplex-real time PCR analysis followed by melting curve analysis offered rapid, sensitive, and specific detection of pork and chicken in sausage products.