Duplex Real-time PCR Research Articles

Detection of toxin-producing Corynebacterium diphtheriae from throat swabs of diphtheria patients using duplex real-time PCR.

Background and Objectives:Diphtheria is a potentially fatal disease caused by toxigenic bacterial infection, particularly from Corynebacterium diphtheriae (C. diphtheriae). Isolation of C. diphtheriae is technically lacking in sensitivity, and Elek’s test to detect toxin production has several difficulties associated with its application. Duplex real-time PCR to throat swab of suspected diphtheria patients can detect both bacteria and toxin-encoding genes simultaneously, faster, with higher sensitivity and specificity.Materials and Methods:A total of 89 consecutive throat swabs from suspected diphtheria patients were collected from Sulianti Saroso Infectious Disease Hospital, Jakarta, during 2018 to 2019. Two pairs of primers and probes, targeting the rpoB gene of C. diphtheriae and the A-subunit of the diphtheria toxin gene, were used in this study. Parameters including annealing temperature, concentration of primers and probes, inhibitors, cross-reaction and detection limit were all optimized. Elek’s toxigenicity test and clinical data were analyzed for comparison.Results:The optimum annealing temperature was 55°C. The concentrations of Cd primer, Tox primer, Cd probe and Tox probe were 0.4, 0.6, 0.5 and 0.625 µM, respectively. DNA elution and template volumes were 50 µL and 5 µL. The detection limit was 2 CFU/mL. No cross-reaction with other microorganisms was observed. Of the 89 samples, duplex real-time PCR gave better results than the standard test, with 19 (21.3%) and 10 (11.2%) patients diagnosed with diphtheria, respectively.Conclusion:Duplex real-time PCR increases the rate of laboratory diagnosis of diphtheria, compared to the standard method to detect potentially toxigenic C. diphtheriae.

Specific Detection and Differentiation Between Brucella melitensis and Brucella abortus by a Duplex Recombinase Polymerase Amplification Assay.

Brucellosis is a highly contagious zoonosis caused by a species under the genus Brucella. A duplex recombinase polymerase amplification (Duplex RPA) assay for the specific detection of Brucella melitensis and Brucella abortus was developed in this study. Primers were designed targeting hypothetical protein genes and membrane transporter genes of B. melitensis and B. abortus, respectively. The newly developed assay was validated for its analytical sensitivity and specificity. Different samples were collected from the Qinghai, Inner Mongolia, and Xinjiang provinces. After DNA extraction, the samples were analyzed by Duplex RPA, real-time PCR, and multiplex AMOS PCR to estimate the prevalence of brucellosis in sheep and yak in West China. The analytical sensitivities of Duplex RPA were 9 × 102 plasmid copies of B. melitensis and 9 × 101 plasmid copies of B. abortus, but by mixing the reaction tubes after 4 min of incubation, the sensitivities were 4 × 100 and 5 × 100 copies of B. melitensis and B. abortus, respectively. There was no cross-reactivity with Brucella suis, Chlamydia abortus, Salmonella typhimurium, Escherichia coli, and Toxoplasma gondii. The screening of field samples by Duplex RPA revealed that the prevalence of B. melitensis in sheep and yak was 75.8% and the prevalence of B. abortus was 4.8%. Multiplex AMOS PCR showed that the prevalence of B. melitensis was 19.3%, and that of B. abortus was 4.8%. It was concluded that the developed Duplex RPA is sensitive and specific to the detection of and differentiation between B. melitensis and B. abortus which will be useful in epidemiological surveillance and in the clinical settings.

Food fraud in the Alps? — Detection of chamois (Rupicapra rupicapra) in firm raw sausages, ham, and meat via qualitative duplex real-time PCR

BackgroundFirm raw sausages or ham from game species like chamois, red deer, or roe deer are typical meat products from the alpine regions in Germany, Italy, Austria, and Switzerland. Increased hunting effort and limited numbers make chamois meat more expensive than comparable game meat. In routine analysis at Bavarian Health and Food Safety Authority (LGL), screening of meat relevant animal species is usually performed by LCD array. However, chamois is not included on the commercial array. A simple alternative method for chamois detection in mixed products was therefore required. ResultsWe developed a duplex probe-based real-time PCR (duplex qPCR) that facilitates the specific detection of chamois and a universal eukaryotic gene as control, especially in processed meat products. Detection of chamois was sensitive (chamois DNA content of 1.25 pg/μL or 0.05% is detectable), robust (eight variations of PCR conditions tested), and specific (inclusivity and exclusivity). Detection of Eukarya in the duplex assay can be used both as an inhibition control, and as a rough estimation for the chamois content in the sample. We analysed 20 firm raw sausages with chamois as a declared ingredient. Eleven sausages were free of chamois meat, for three sausages, a ΔCq(Chamois-Eukarya) > 10 indicated the use of chamois meat as minor ingredient, and in six sausages, abundant amounts of chamois meat were detected. Furthermore, among three analysed chamois hams, one consisted of roe deer meat instead. ConclusionsThe developed duplex qPCR is an easy to use tool to verify the presence of the declared ingredient chamois meat, especially in processed meats like sausages. The considerable proportion of products lacking declared chamois meat (55% or 11/20 for firm raw sausages, 33% or 1/3 for ham), indicates the need for further controls in this non-standard food segment.

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.

Applicability of a duplex and four singleplex real-time PCR assays for the qualitative and quantitative determination of wild boar and domestic pig meat in processed food products

Appropriate analytical methods are needed for the detection of food authentication. We investigated the applicability of a duplex real-time PCR assay targeting chromosome 1 and two singleplex real-time PCR assays targeting chromosome 9, both published recently, for the qualitative and quantitative determination of wild boar and domestic pig in processed food products. In addition, two singleplex real-time PCR assays targeting chromosome 7 were tested for their suitability to differentiate the two subspecies. Even by targeting the three genome loci, the probability of misclassification was not completely eliminated. Application of the real-time PCR assays to a total of 35 commercial meat products, including 22 goulash products, revealed that domestic pig DNA was frequently present, even in 14 out of 15 products declared to consist of 100% wild boar. Quantitative results obtained with the real-time PCR assays for wild boar (p < 0.001) and those for domestic pig (p < 0.001) were significantly different. However, the results obtained with the real-time PCR assays for wild boar (r = 0.673; p < 0.001) and those for domestic pig (r = 0.505; p = 0.002) were found to be significantly correlated. If the rules given in the paper are followed, the real-time PCR assays are applicable for routine analysis.

Development and validation of a duplex real-time PCR assay with locked nucleic acid (LNA) probes for the specific detection of allergenic walnut in complex food matrices

Walnut is one of the most widely consumed and highly allergenic tree nuts in North America. The two species most frequently consumed in the United States are English walnut (Juglans regia) and black walnut (Juglans nigra). Ensuring accurate labeling to protect allergic individuals from accidental consumption demands methods that accurately identify the presence of walnut in food. We developed a novel real-time PCR assay that targets two chloroplast regions (rbcL-accD and psbM-trnD) and implements LNA probes to specifically detect walnut in three types of foods: baked goods, salad greens, and creamy dips. This is the first assay for walnut detection that uses LNA oligos to increase specificity and prevent significant cross-reactivity with pecan and other tree nuts by blocking their amplification signal. Food matrices were spiked separately with English and black walnut at concentrations that ranged from 0.1 to 105 mg/kg (parts per million, or ppm). Reaction efficiencies within the optimal range of 100 ± 10% were obtained across 6 to 7 orders of magnitude with a limit of detection of 0.1 ppm. Our results show that real-time PCR assays using chloroplast markers and LNA probes are an effective and accurate tool for allergenic walnut detection.

Duplex real-time PCR for sexing Schistosoma japonicum cercariae based on W chromosome-specific genes and its applications.

As a unique feature among otherwise hermaphroditic trematodes, Schistosoma species are gonochoric parasites whose sex is genetically determined (ZZ for males and ZW for females). However, schistosome larvae are morphologically identical, and sex can only be discriminated by molecular methods. Here, we integrated published Schistosoma. japonicum transcriptome and genome data to identify W chromosome-specific genes as sex biomarkers. Three W chromosome-specific genes of S. japonicum were identified as sex biomarkers from a panel of 12 genes expressed only in females. An efficient duplex real-time PCR (qPCR) method for sexing cercariae was developed which could identify the sex of cercariae within 2 h without DNA extraction. Moreover, this method can be used to identify not only single-sex but also mixed-sex schistosome-infected snails. We observed a nearly equal proportion of single-male, single-female, and mixed-sex schistosome infections in artificially infected snails. Sex-known schistosome-infected snail models can be efficiently constructed with the aid of duplex qPCR. A field study revealed that single-sex schistosome infections were predominant among naturally infected snails. Finally, a schistosomiasis mouse model based on sex-known cercariae infection was shown to be more reliable than a model based on sex-unknown cercariae infection. The developed duplex qPCR method for sexing S. japonicum cercariae can be widely used for schistosomiasis modeling, genetic experiments, and field-based molecular epidemiological studies.

Cardiopulmonary nematodes of wild carnivores from Denmark: Do they serve as reservoir hosts for infections in domestic animals?

The cardiopulmonary nematodes Angiostrongylus vasorum, Crenosoma vulpis, Capillaria aerophila and Aelurostrongylus abstrusus, are a cause of concern in the scientific and veterinary community, potentially causing significant disease in domestic animals. To investigate the potential of wild carnivores as reservoir hosts to these parasites, a total of 1041 animals from seven regions of Denmark were sampled: 476 raccoon dogs (Nyctereutes procyonoides), 367 red foxes (Vulpes vulpes), 123 American mink (Neovison vison), 31 beech martens (Martes foina), 30 Eurasian otters (Lutra lutra) and 14 polecats (Mustela putorius). Hearts and lungs were collected and examined for cardiopulmonary parasites. Capillaria aerophila was identified using morphology, whereas A. vasorum and C. vulpis were identified by a duplex real-time PCR, and A. abstrusus by conventional PCR. This is the first Danish report of A. vasorum and C. vulpis infections in raccoon dogs, mink and polecats, and of C. aerophila in raccoon dogs and beech martens. In addition, this is the first time A. vasorum and C. vulpis have been identified in wild animals from the island of Bornholm, just as it is the first report of C. vulpis in American mink, and C. vulpis and A. vasorum in polecats in Europe. The prevalence of A. vasorum appears to have increased in red foxes in Denmark compared to previous studies, while C. vulpis and C. aerophila prevalences are lower. Our data show that several wild carnivores can serve as reservoir hosts for A. vasorum, C. vulpis and C. aerophila in Denmark, and that A. vasorum appears more abundant than previously reported. It is speculated that the A. vasorum increase might relate to increased snail abundance, which may be due to a rise in mean yearly temperatures in Denmark.

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

Powdery mildew and downy mildew are two devastating diseases on cucumber and other cucurbit crops caused by Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Identification and detection of these pathogens from field and plant material could be significant for the selection of resistant varieties and formulation of disease management strategies. In the present study, a duplex qPCR assay developed for simultaneous detection and quantification of both pathogens from different samples. Two sets of species-specific primers developed for the detection of P. xanthii and P. cubensis pathogens by targeting the internal transcribed spacer (ITS) region of the rDNA gene cluster. The specificity of designed primers was also evaluated against the different microbial, plant, soil, and environmental samples. Initially, the individual assays for P. cubensis and P. xanthii were validated using their corresponding species-specific primers, which amplified the prominent and distinctive products of ~ 705 bp and ~ 290 bp size, respectively. SYBR green-based duplex real-time PCR assay was developed to detect and quantify both mildew pathogens from different field samples. The species-specific oligonucleotide primer sets showed high specificity with melt curve peaks at 85.83 °C and 88.05 °C, for P. xanthii and P. cubensis, respectively. The relative quantification and lowest detection limit of qPCR assays using tenfold diluted plasmid (Csp1 and Csd1) DNA were estimated (0.1 pg/µl) through a standard curve. In this study, the species-specific PCR and qPCR assays in both simplex and duplex formats have been validated successfully. These assays could be useful for efficient detection and quantification of mildew pathogens from the cucumber and other cucurbit crops.

Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

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.

Development of a Duplex Real-time PCR for Differentiation of Salmonella Typhimurium and Monophasic Serovars

Development of a Duplex Real-time PCR for Differentiation of Salmonella Typhimurium and Monophasic Serovars

Hyperendemic Dirofilaria immitis infection in a sheltered dog population: an expanding threat in the Mediterranean region

A study on the occurrence of Dirofilaria immitis and its vectors was carried out in order to assess the prevalence of the disease in dogs in previously non-endemic areas of southern Italy. Blood samples (n = 385) and mosquitoes (n = 1540) were collected in two dog shelters and analysed by Knott’s test and duplex real-time PCR, respectively. Dirofilaria immitis was the most prevalent filarioid (44.2%), while Culex pipiens was the most prevalent mosquito species (68.8%). This high prevalence of D. immitis infection confirms this location as one of the most hyperendemic foci of dirofilariosis in Europe.

Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/μL, 30.6 copies/μL and 60 copies/μL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.

Duplex qPCR assay for detection and quantification of Anaplasma phagocytophilum and Rickettsia spp.

Anaplasma phagocytophilum and Rickettsia spp. are vector-borne zoonotic bacteria, which are clinically important especially in immunocompromised patients. There are large gaps in the current knowledge of their geographic distribution and prevalence in both their vectors and hosts. Our aim was to develop reliable and easy detection method for both these pathogens. We made a new hydrolysis probe based duplex Real-Time PCR assay based on previous studies. We optimized the assays and tested them to provide reliable recommended procedures with a sensitivity to a minimum of 10 target DNA copies per sample. The assays were designed to be specific for A. phagocytophilum and in the same reaction detect multiple species of rickettsiae. We designed gBlock quantification standards that provide the option to identify differences in pathogen load among different samples in subsequent studies.

Duplex real-time quantitative PCR for simultaneous detection and quantification of Pratylenchus neglectus and P. thornei

Pratylenchus neglectus and P. thornei are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the P. neglectus-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the P. thornei-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other Pratylenchus species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from P. neglectus and P. thornei DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (Ct) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of P. neglectus and P. thornei.

Evaluation of different methods for preventing the African swine fever virus

African swine fever (ASF) is a contagious disease of key importance that occurs in wild boar population and domestic pigs. To date, there is no vaccine or treatment available, hence disease surveillance and stamping out in case of an outbreak is the only way to control it. A duplex ASF-IC real-time PCR assay was optimised using field samples obtained in Lithuania and results of singleplex and duplex real-time PCR assays allowing the identification of the ASF virus were compared. The specificity and sensitivity of ASF virus detection was validated using a reference sample of positive and negative virus nucleic acid and ASF virus positive and negative samples obtained from animals in Lithuania. Results prove that duplex ASF-IC real-time PCR assay provides a rapid, sensitive, and reliable molecular tool for ASF virus detection in wild boar population and domestic pigs. In general, it was found that duplex ASF-IC real-time PCR assay proves to be faster and more sensitive than singleplex real-time PCR and therefore has a high potential to be applied in routine analysis.

Development of a duplex SYBR GreenⅠ based real-time PCR assay for detection of porcine epidemic diarrhea virus and porcine bocavirus3/4/5

The duplex real-time PCR assay based on SYBR Green І was developed for detection of porcine epidemic diarrhea virus (PEDV) and porcine bocavirus (PBoV) 3/4/5 genotypes simultaneously. Two pairs of specific primers were designed targeting the N gene sequence of PEDV and VP1 gene sequence of PBoV3/4/5. PEDV and PBoV3/4/5 could be distinguished by their different melting temperatures (Tm) in one sample. The Tm value of PEDV was 83.5 °C, and the Tm value of PBoV3/4/5 was 78.5 °C, while other swine pathogens showed no specific melting peaks. The detection limits of this assay were 10 copies/μL for both PEDV and PBoV3/4/5. A total of sixty-three intestinal tissue samples were collected from piglets suffering from diarrhea, and the viral nucleic acids detected and identified by the real-time PCR assay and conventional PCR assay. The duplex real-time PCR detection results showed that the prevalence of PEDV and PBoV3/4/5 was 85.7% and 46%, respectively, and the co-infection rate of the two viruses was 28.6%. These results indicated that this duplex real-time PCR assay was a sensitive, specific and reproducible method for differentiating PEDV and PBoV3/4/5 or their co-infection.

Detection of Ehrlichia spp. and Anaplasma phagocytophilum in whole blood specimens using a duplex real-time PCR assay on the ARIES instrument

Cases of tick-borne diseases are increasing in the United States, and new tick-borne pathogen species causing human illness are being discovered. The specific etiology is generally difficult to diagnose based on clinical signs and symptoms alone, because of their generalized nature and often lack of a known tick bite. For some infections, such as Lyme disease and spotted fever group rickettsioses, serology remains the most appropriate laboratory diagnostic tool, but for others such as anaplasmosis, ehrlichiosis, and babesiosis, direct detection in the blood is preferred for rapid diagnosis. In Kentucky, USA, the area served by our laboratory, the most commonly reported tick-borne illnesses include spotted fever group rickettsiosis, ehrlichiosis and Lyme disease, but of these three diseases, only ehrlichiosis is well-suited for direct detection using PCR methods during the acute stage of illness. We report here the validation of a duplex real-time PCR assay using whole blood specimens on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. This method allows for rapid and sensitive detection of acute infections with Ehrlichia spp. and Anaplasma phagocytophilum using whole blood specimens. We included A. phagocytophilum to monitor emergence of this pathogen in Kentucky, since surrounding states have reported many more cases than Kentucky.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/μL for CSFV and 36 copies/μL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.