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Porcine circovirus type 3 (PCV3) is a novel pathogen first identified in the United States in 2016. As there is a high possibility that no clinical signs of infection are observed in the host, an accurate and sensitive method is needed for quarantine on numerous live pigs especially for international pig trade. In this study, a TaqMan‐based real‐time PCR assay specifically for PCV3 was established without cross‐reactions with other non‐targeted pig viruses. The sensitivity of the current approach is about 1.5 × 101 copies μL−1 plasmid DNA while the sensitivity of the conventional PCR is about 1.5 × 102 copies μL−1 plasmid DNA. Further, this assay was applied in the retrospective quarantine on serum samples of 601 commercial live boars imported to China from the United States, France and the United Kingdom from 2011 to 2017. The results revealed that PCV3 could be detected positive in the commercial boars imported from the United States and the above‐mentioned western European countries and phylogenetic study also revealed that viral isolates were grouped with some isolates from Korea and the United States. Our study suggested that PCV3 may be prevalent globally since 2011. In this study, we developed a real time PCR assay for PCV3 detection. Using this assay, It has been demonstrated for the first time the detection of PCV3 in UK, France (and in USA, China) in samples collected between 2011 and 2017.

Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost–benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqMan® qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used.

Background Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. Results After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/μl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A. , S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. Conclusions We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

Background Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV. Results The specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/μl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings. Conclusions Our data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.

Introduction: As the most reliable means of diagnosing hepatitis (HBV) infection and predicting the prognosis of HBV-related chronic liver disease, the COBAS AmpliPrep/COBAS TaqMan real-time polymerase chain reaction (PCR) (CAP/CTM) assay provides a highly sensitive and accurate method for quantifying HBV DNA. However, the high cost of the COBAS reagents is prohibitive in many developing countries. Thus, we compared the Sansure magnetic bead (Magb) assay, a novel technology developed by a Chinese company, with the CAP/CTM assay. Methodology: The reproducibility and sensitivity of the Sansure Magb assay were first validated using HBV DNA reference samples. Next, the quantitative results for the two assays using 635 blood samples collected from chronic hepatitis B patients and 10 healthy controls were compared. Results: The Sansure Magb assay showed high reproducibility and was at least as sensitive and specific as the CAP/CTM assay. Among the patient samples, 407 tested positive by both methods, with 386 (94.84%) showing quantitative differences of less than 1 log unit and 21 (5.16%) showing quantitative differences of between 1 and 2 log units. The results from the assays were closely correlated. Bland-Altman plot analysis showed that only 6.6% of the data points fell outside the 95% limits of agreement, which suggests that the differences between methods are clinically acceptable. Conclusions: This study demonstrates that the Sansure Magb assay is highly sensitive and reproducible. Based on its reduced cost, the Sansure Magb assay may be more applicable than the CAP/CTM assay for HBV diagnosis in developing countries such as China.

Stomach contents of potential predators from a flatfish nursery ground on the Scottish west coast were examined visually and probed using a TaqMan real-time PCR based assay designed to detect plaicePleuronectes platessaDNA. Stomachs from 1137 brown shrimpCrangon crangon, along with a lesser number of shore crabCarcinus maenas, grey gurnardEutrigla gurnardusand Gobiidae were analysed. Overall 45% of shrimp tested positive for plaice DNA, a proportion considerably higher than in stomachs containing visually identifiable flatfish remains. When scaled to the population level, predation by shrimp generated an estimated mortality of ~9% d–1, which compares with a decline in plaice abundance from mid-May to mid-June of ~4.4% d–1. The discrepancy between mortality estimates based on molecular and catch-curve analysis might be due to sampling being conducted at low water between dusk and dawn, which would concentrate predators and prey at times coincident with peaks of shrimp feeding. In addition, the sensitivity of the TaqMan assay could have led to some over-estimation because non-fatal interactions may also have been detected, e.g. shrimp are known to nibble the fins of flatfish, that might result in a positive TaqMan result but not necessarily in mortality for the fish. The percentage of shrimp testing positive for presence of plaice DNA in their stomachs was also related to plaice density in a positive, linear manner. For less abundant predators, stomachs of 6% of shore crabs, 40% of gurnards and 11% of large gobies also tested positive.

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.

A conventional PCR (cPCR) remains an effective molecular technique for the diagnosis of canine monocytic ehrlichiosis. However, agarose gel electrophoresis requires additional time after thermal cycling. In the present study, we developed a PCR-based lateral flow biosensor (PCR-LFB) to detect Ehrlichia canis (E. canis). Lateral flow strips allow for the simple and rapid detection of PCR products and provide an alternative to gel electrophoresis. The sensitivity, specificity, and detection limit of PCR-LFB were compared to those of TaqMan probe-based real-time PCRs (qPCRs). The PCR-LFB was performed with 5′ 6-FITC and biotin-labeled primers specific to E. canis, targeting the dsb gene. The detection limit of the PCR-LFB assay was 10−6 for the target DNA sequence in a 10-fold dilution of the recombinant plasmid, which is 10 times lower than that of qPCR. Among the confirmed qPCR results in the 30 dog samples, false-positive results were not detected by the PCR-LFB. Compared to qPCR, the sensitivity and specificity of PCR-LFB were 63.6% (95% CI; 42.9–80.2%) and 100% (95% CI; 67.5–100%), respectively. The Kappa value of the PCR-LFB is in moderate agreement with the qPCR (κ = 0.483). Perfect agreement (κ = 1) was observed between cPCR and PCR-LFB. Lower cost and shorter time consumption were demonstrated using PCR-LFB.

Background In Ghana, pre-school-aged children (PSAC) are at risk of intestinal schistosomiasis and are living in need of praziquantel treatment. To better assess the infection burden within this vulnerable demographic group, we have provided a comparative assessment of the prevalence of Schistosoma mansoni in pre-school-aged children by urine circulating cathodic antigen (CCA) dipsticks, real-time PCR Taqman® faecal assays and Kato-Katz coproscopy. Methods In all, 190 pre-school-aged children were sampled from three endemic communities ( viz . Tomefa, Torgahkope/Adakope, and Manheam) around Weija dam, Southern Ghana. Fresh stool and urine samples were collected from all participants for diagnosis. Results Among all the three communities, the urine-CCA assay recorded the highest prevalence values of 90.5% (95% CI 80.4–96.4), 87.9% (95% CI 76.7–95), and 81.2% (95% CI 69.9–89.6) in Tomefa, Torgahkope/Adakope, and Manheam respectively. Prevalence by real-time PCR was 50% (95% CI 35.5–64.5), 8% (95% CI 2.2–19.2) and 16.7% (95% CI 8.3–28.5), while by Kato-Katz was 55.6% (95% CI 42.5–68.1), 8.6% (95% CI 2.9–19) and 11.6% (95% CI 5.1–21.6) respectively. Children aged 1 year and over were found to be positive with the urine-CCA assay; by the ages of 3–4, over 50% were urine-CCA patent. The sensitivity and specificity of the POC-CCA dipsticks, when compared against the combined results of Kato-Katz/TaqMan results was found to be 84.1% (95% CI = 72.7–92.1) and 12.9% (95% CI = 6.6–22) respectively. Conclusions We propose that the urine-CCA dipstick may be a useful rapid diagnostic tool to estimate the prevalence of intestinal schistosomiasis in PSAC, particularly in rapid identification of at-risk areas. However, our assessment has shown that it possible to record false positives when compared to combined Kato-Katz and qPCR results. To guide PSAC praziquantel treatment needs, we propose the urine CCA assay should be included in routine surveillance of intestinal schistosomiasis alongside other diagnostics such as Kato-Katz and urine filtration.

Background In China, Newly emerging duck reovirus (NDRV) variants have been causing major disease problems in cherry valley ducks. NDRV has the potential to cause high morbidity and 5–50% mortality rates. Severe hemorrhagic-necrosis in the liver and spleen were commonly seen in NDRV affected ducks. The availability of upgraded methods for rapid diagnosis of newly emerging DRV variants is crucial for successful DRV infection control and prevention. Results In this study, we present a TaqMan-based real-time PCR assay (RT-qPCR) for the detection of NDRV infection. Using the conserved regions within the NDRV genome, we designed the specific primers and probe. The lower limit of detection for NDRV infection was 10 copies/μL (Ct values: 38.3) after the optimization of the RT-qPCR conditions. By cross-checking with other duck viral pathogens, no cross-reactivity was observed confirming the assay was highly specific for the detection of NDRV. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assay variability was less than 2.91%(Intra-assay variability of Ct values: 0.07–1.48%; Interassay variability of Ct values: 0.49–2.91%). This RT-qPCR and conventional PCR (cPCR) detected one hundred and twenty samples of NDRV infection from different regions. The result shows that the positive rates were 94.17 and 84.17% respectively. The detection rate of RT-qPCR rapid detection assay was 10% higher than that of the cPCR method. Conclusion This research developed a highly sensitive, specific, reproducible and versatile of RT-qPCR for quantitatively detecting NDRV. It can be used to study the pathogenesis and epidemiology investigation of NDRV.