TaqMan-based Real-time PCR Assay Research Articles

Active Surveillance of Powassan Virus in Massachusetts Ixodes scapularis Ticks, Comparing Detection Using a New Triplex Real-Time PCR Assay with a Luminex Vector-Borne Panel.

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon-exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha's Vineyard Island in Massachusetts. The assay's performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems.

Application and development of a TaqMan-based real-time PCR assay for rapid detection of snakehead vesiculovirus.

Snakehead vesiculovirus (SHVV) is one of the primary pathogens responsible for viral diseases in the snakehead fish. A TaqMan-based real-time PCR assay was established for the rapid detection and quantification of SHVV in this study. Specific primers and fluorescent probes were designed for phosphoprotein (P) gene, and after optimizing the reaction conditions, the results indicated that the detection limit of this method could reach 37.1 copies, representing a 100-fold increase in detection sensitivity compared to RT-PCR. The specificity testing results revealed that this method exhibited no cross-reactivity with ISKNV, LMBV, RSIV, RGNNV, GCRV, and CyHV-2. Repetition experiments demonstrated that both intra-batch and inter-batch coefficients of variation were not higher than 1.66%. Through in vitro infection experiments monitoring the quantitative changes of SHVV in different tissues, the results indicated that the liver and spleen exhibited the highest viral load at 3 poi. The TaqMan-based real-time PCR method established in this study exhibits high sensitivity, excellent specificity, and strong reproducibility. It can be employed for rapid detection and viral load monitoring of SHVV, thus providing a robust tool for the clinical diagnosis and pathogen research of SHVV.

Application of quantitative real-time PCR to detect Mink Circovirus in minks, foxes and raccoon dogs in northern China.

Mink circovirus disease caused by Mink Circovirus (MiCV) is a serious infectious disease of mink that has become prevalent in recent years in China, severely affecting the reproductive performance of mink and causing significant economic losses to farms. To date, there have been few studies on MiCV, its pathogenic mechanism is not clear, and there is no effective vaccine or drug to prevent and control the disease. Therefore, it is necessary to establish a rapid and reliable molecular diagnostic method, which would aid future studies of this novel virus. In our study, we developed a sensitive and specific TaqMan-based quantitative real-time PCR assay targeting the MiCV Cap gene. The assay showed no cross-reaction with other tested animal viruses. The assay is highly sensitive, with a detection limit of as low as 10 plasmid DNA copies and 2.38 × 10-2 pg of viral DNA. The intra and inter--assay coefficients of variation were both low. The positive detection rate of MiCV in clinical samples from minks, foxes, and raccoon dogs were 58.8% (133/226), 50.7% (72/142), and 42.2% (54/128), respectively, giving a total positive detection rate of 52.2% (259/496). Higher contamination levels were observed in samples from the environment in direct or indirect contact with animals, with a total positive detection rate of 75.1% (220/293). These epidemiological results showed that minks, foxes, and raccoon dogs had high infection rates of MiCV. This was also the first study to detect MiCV on the ground and equipment of fur-bearing animal farms. Our assay is highly sensitive and specific for the diagnosis and quantification of MiCV, and should provide a reliable real-time tool for epidemiological and pathogenetic study of MiCV infection.

Development and application of a TaqMan-based real-time PCR method for the detection of the ASFV MGF505-7R gene.

African swine fever virus (ASFV), the etiological agent of African swine fever (ASF), causes deadly hemorrhagic fever in domestic pigs. ASF's high mortality and morbidity have had disastrous effects on the world's swine industry. In recent years, the number of African swine virus strains has increased and presented new challenges for detecting classical ASFV-p72-based viruses. In this study, we observed that the ASFV MGF505-7R gene, a member of the multigene family that can enhance ASFV virulence and pathogenesis, has the potential to be a candidate for vaccine formulations. We also developed a real-time PCR assay based on the ASFV MGF505-7R gene and validated it in multiple aspects. The results indicated that the approach could detect standard plasmids with a sensitivity and a specificity of up to 1 × 101 copies/μL. Moreover, the assay had no cross-reactions with other porcine viruses. In laboratory and clinical settings, the assay can detect ASFV-infected samples at an early stage (4 hpi) and show a consistency of 92.56% when compared with classical ASFV detection in clinically ASFV-infected materials. This study's results also indicated that the TaqMan-based quantitative real-time PCR assay we developed for detecting the ASFV MGF505-7R gene is both sensitive and specific. This assay can provide a quick and accurate method for detecting ASFV and has the potential to be used as an optional tool for screening and monitoring ASF outbreaks.

Development of TaqMan-based real-time PCR assay based on the E1 genefor the quantitative detection of the Getah virus.

To develop a sensitive, specific, and rapid approach for the detection Getah virus (GETV), a set of primers targeting the conserved region of the E1 gene was created. The TaqMan-based real-time PCR method for GETV detection was developed by optimizing the reaction conditions. The method demonstrated excellent specificity, and amplification did not occur with the causative agents of all prevalent swine viral infections (CSFV, PRRSV, PRV, PEDV, PTV, and JEV), except GETV. Additionally, upon assessing the sensitivity of the method, the minimum detection limit for GETV was found to be 5.94 copies/μL, which is 10 times higher than that of the traditional PCR approach. Further, the intra- and inter-assay variation coefficients were less than 1%, demonstrating good repeatability. Moreover, GETV was found in 10 of the 20 field serum samples using real-time PCR but only in three of the samples using traditional PCR. Consequently, the first GETV TaqMan-based real-time PCR approach based on the E1 gene was developed for GETV pathogenic diagnoses, and this exhibited high specificity, sensitivity, and repeatability. This assay is practical for the pathogenic diagnosis and epidemiology of GETV.

Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi.

Salmonella enterica subsp. enterica serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect S. Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqMan-based real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/μL of standard plasmid and 10 CFU/μL of bacterial DNA. Furthermore, 540 clinical samples, including 162 tissue, 192 plasma, and 186 vaginal swab samples collected between 2018 and 2021 in China, were tested to assess the performance of the developed assay. Compared to the gold standard method of bacterial isolation, the real-time PCR assay exhibited 100% positive agreement for all tissue, plasma and vaginal swab tests. Additionally, this assay detected DNA from S. Abortusequi from 56.7% (34/60) culture-negative tissue and 22.9% (41/179) culture-negative vaginal swab samples from infected equids. Receiver operating characteristic analysis demonstrated that the results of the developed real-time PCR assays were in significant agreement with those of the culture method. The real-time PCR assay can be completed within 45 min of extraction of DNA from samples. Our results show that this assay could serve as a reliable tool for the rapid detection of S. Abortusequi in tissue, plasma, and vaginal swab clinical samples.

Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

Simple SummaryAmerican foulbrood (AFB) is the most severe bacterial disease of honeybees, caused by Paenibacillus larvae. Larvae become infected by ingesting food contaminated with P. larvae spores, which are extremely resistant and can remain infectious for decades. Burning affected colonies is widely used to prevent further spread of the disease. The presence of P. larvae spores in bee-related samples is associated with an increased risk of developing clinical symptoms, and spore counts can be used for early detection of at-risk colonies, which should then undergo thorough clinical examination. Because quantification of P. larvae spores by plate counting is time-consuming and unreliable, due to poor and inconsistent germination, molecular quantification is more suitable. To overcome the limitations of available quantification methods, we developed a quantitative PCR (qPCR) assay for reliable quantification of P. larvae that also performs well at low spore counts. The assay was validated for honey and hive debris samples but can be extended to other sample types. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies, both for honey and hive debris samples. By comparing plate and qPCR counts, the germination rate of P. larvae spores was found to be low and inconsistent.Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

Establishment of TaqMan-based real-time PCR assay for rapid detection of duck circovirus.

Duck circovirus (DuCV) is widespread across the world and causes feather disorders in young ducks. It was identified as the causative pathogen of duck beak atrophy and dwarfism syndrome and primary sclerosing cholangitis. In this study, we aimed to establish a TaqMan-based real-time PCR assay to detect DuCV. The primers and probe were designed based on the conserved region of the DuCV Rep gene. After optimizing the reaction conditions, the minimum virus detection limit of the designed PCR technique was 39.4 copies/μL, 100 times that of conventional PCR (cPCR). No cross-reaction with six other common duck viruses was observed. The intra- and inter-assay variations were less than 1%. The detection rate of DuCV-positive clinical samples using TaqMan-based real-time PCR was higher than that using SYBR Green-based real-time PCR and cPCR. Collectively, these results showed that the established TaqMan-based real-time PCR detected DuCV with high sensitivity and specificity, and significant repeatability, making it suitable for clinical use. Hence, it may be used as a novel tool for the diagnosis and epidemiological investigation of DuCV.

Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL−1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL−1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL−1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray’s Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

Development and application of a TaqMan-based real-time PCR assay for specifically detecting feline astrovirus

Feline astrovirus (FeAstV), an enteric RNA virus of recent concern that is associated with diarrheal illness in cats, has been described in several countries throughout the world. However, no scientific and sensitive diagnostic method against FeAstV was reported up to now. Here, we developed a specific, sensitive and repeatable TaqMan fluorescence quantitative PCR (qPCR) assay to investigate the prevalence of FeAstV in domestic cats from China, especially low copy numbers in clinical sample. Specific assay showed that no cross-reactivity was observed with other non-FeAstV cat-derivied pathogens, suggesting this method was highly specific for FeAstV. The lowest detection limit of this assay was 3.52 copies/μl, and 1000-times more sensitive than conventional PCR. Intra- and inter-assay variability was less than 1.72%, means a high degree of repeatability. A total of 578 clinical fecal samples were collected from northeast China, and were tested for FeAstV using our developed qPCR assay. 105 samples were positive for FeAstV with an overall prevalence of 18.17%. Moreover, a higher positive rate was found in cats with diarrhea (32.26%, 80/248) than that in asymptomatic cats (7.58%, 25/330), further demonstrating that FeAstV infection was associated with diarrhea in cats. In brief, our developed assay showed high specificity, sensitivity, reproducibility for detecting FeAstV, and can be used for clinical diagnosis and epidemiological investigation of FeAstV.

Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of Vibrio cholerae and Vibrio parahaemolyticus on the BD MAX Platform.

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay

H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was recently discovered and proposed to cause runting-stunting syndrome and urate deposition in geese. At present, however, there is a lack of epidemiological information pertaining to the dynamics and distribution of H146-like GCV. The development of novel molecular diagnostic approaches capable of rapidly and accurately detecting this virus would support the strengthening, the prevention, and control of H146-like GCV infection. In the present study, we therefore used a TaqMan probe and primers specific for the viral nonstructural (NS) gene to develop a highly sensitive and specific PCR assay capable of detecting this H146-like GCV. The assay reproducibly detected 5.07 × 102 copies of a recombinant DNA plasmid containing the NS gene, with a dynamic range of 8 orders of magnitude (102–109 copies). Importantly, no cross-reactivity was observed with common viruses that affected waterfowl, and when we used this assay to evaluate clinical samples, we found it to be more sensitive and faster than traditional PCR. In summary, herein, we developed a novel TaqMan-based real-time PCR approach that could reliably detect and diagnose H146-like GCV. This tool will allow for the real-time diagnosis of H146-like GCV infections, enabling researchers to better understand the epidemiology and clinical presentation of this disease.

A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China

BackgroundIn 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.ResultsIn 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.ConclusionThis 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.

A TaqMan-based multiplex real-time PCR assay for the rapid detection of tigecycline resistance genes from bacteria, faeces and environmental samples

BackgroundTigecycline is a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant bacteria. Recently, novel tigecycline resistance genes tet(X3) and tet(X4) have been reported, which pose a great challenge to human health and food security. The current study aimed to establish a TaqMan-based real-time PCR assay for the rapid detection of the tigecycline-resistant genes tet(X3) and tet(X4).ResultsNo false-positive result was found, and the results of the TaqMan-based real-time PCR assay showed 100% concordance with the results of the sequencing analyses. This proposed method can detect the two genes at the level of 1 × 102 copies/μL, and the whole process is completed within an hour, allowing rapid screening of tet(X3) and tet(X4) genes in cultured bacteria, faeces, and soil samples.ConclusionTaken together, the TaqMan-based real-time PCR method established in this study is rapid, sensitive, specific, and is capable of detecting the two genes not only in bacteria, but also in environmental samples.

Development of a TaqMan-based real-time PCR assay for detection and quantification of Pythium aphanidermatum in plant and soil samples

ABSTRACT The soilborne oomycete pathogen Pythium aphanidermatum causes damping-off and root rot in a wide range of economically important crops and vegetables. Effective P. aphanidermatum disease management is hindered by the lack of accurate pathogen detection methods. We developed a TaqMan-based real-time PCR tool for efficient detection and quantification of P. aphanidermatum presence in plant and soil samples. Eukaryotic peptide chain release factor subunit 1 (eRF1) was selected as the marker gene based on bioinformatic analysis. P. aphanidermatum DNA was quantified by using a eRF1-containing plasmid as the reference standard. This approach is sensitive enough to detect as low as 10 fg of P. aphanidermatum genomic DNA or 10 copies of reference plasmid. In a specificity evaluation, eight P. aphanidermatum isolates can be precisely detected out of 44 plant pathogen isolates. Moreover, P. aphanidermatum DNA can be detected in plant leaves 10 min after inoculation when there is no symptom appearance at all. Meanwhile, this assay successfully detected P. aphanidermatum zoospores in the soil at the sensitivity level of 100 zoospores/g, a density sufficient for inhibiting seedling growth. Taken together, the real-time PCR tool could be valuable for the early surveillance and quantification of P. aphanidermatum spread in the field.

A TaqMan-based quantitative real-time PCR assay for identification of the goose circovirus

Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.

A highly sensitive internally-controlled real-time PCR assay for mycoplasma detection in cell cultures

Mycoplasma contamination of cell lines is a common occurrence and may affect the cell line behaviour in a variety of ways. Contamination with mycoplasma is usually not obvious so cell lines should be frequently tested. Several commercially available kits for mycoplasma detection exist, however the Ph. Eur. culture method which can take several weeks to yield results is still considered to be the ‘gold standard’ method. There is therefore a need for rapid alternative methods with comparable sensitivity, specificity and species range. Here, we describe an internally-controlled Taqman-based real-time PCR assay for cell culture medium without the need for DNA extraction. The assay can detect less than 10 CFU of the most frequently encountered mycoplasma contaminants in mammalian cell cultures. The validated assay has the potential to be used as a routine test in the production of cell culture-based Biologicals.

Identification of Small Berry Species in Food and Juice Using TaqMan-Based Real-Time PCR

Abstract Background: Public interest is growing for small berries in recent years because they are very delicious, low in energy, and full of bioactive compounds with potential health benefits. Similar to other food products, adulteration of small berry fruit products poses economic and safety problems to consumers. Objective: To protect consumers and regulate the small berry fruit products market, it is necessary to establish a robust method for detecting the authenticity. Methods: In this study, TaqMan-based real-time PCR assay was established for species identification of cranberry, raspberry, and blueberry to ensure authenticity of commercial small berry food products (pulp, dried fruit, fruit juice, jam, and puree). Results: Absolute detection limit was 0.1 pg/μL DNA for raspberries, 1 pg/μL DNA for blueberries, and 10 pg/μL DNA for cranberries. Practical LOD was 0.1% (v/v) for fresh juice. For processed juice, practical LOD was 1% for blueberry and red raspberries, 0.1% for black and yellow raspberries, and 5% for cranberry. Conclusions: The method was shown to be functional and effective to detect the raw material composition of cranberry, raspberry, and blueberry for commercial products. Highlights: TaqMan probe-based real-time PCR methods were designed to identify three small berries (blueberry, raspberry, and cranberry) in berry products. Efficient DNA recovery methods and detection strategy were established to ensure correct and sensitive testing of fresh small berries exhibited a detection limit of 0.1 to 10 pg/μL. The practical minimum detection levels were 0.1 to 5% in fresh and processed juice, including pasteurization and HTHP.

Identification of Small Berry Species in Food and Juice Using TaqMan-Based Real-Time PCR.

Background: Public interest is growing for small berries in recent years because they are very delicious, low in energy, and full of bioactive compounds with potential health benefits. Similar to other food products, adulteration of small berry fruit products poses economic and safety problems to consumers. Objective: To protect consumers and regulate the small berry fruit products market, it is necessary to establish a robust method for detecting the authenticity. Methods: In this study, TaqMan-based real-time PCR assay was established for species identification of cranberry, raspberry, and blueberry to ensure authenticity of commercial small berry food products (pulp, dried fruit, fruit juice, jam, and puree). Results: Absolute detection limit was 0.1 pg/μL DNA for raspberries, 1 pg/μL DNA for blueberries, and 10 pg/μL DNA for cranberries. Practical LOD was 0.1% (v/v) for fresh juice. For processed juice, practical LOD was 1% for blueberry and red raspberries, 0.1% for black and yellow raspberries, and 5% for cranberry. Conclusions: The method was shown to be functional and effective to detect the raw material composition of cranberry, raspberry, and blueberry for commercial products. Highlights: TaqMan probe-based real-time PCR methods were designed to identify three small berries (blueberry, raspberry, and cranberry) in berry products. Efficient DNA recovery methods and detection strategy were established to ensure correct and sensitive testing of fresh small berries exhibited a detection limit of 0.1 to 10 pg/μL. The practical minimum detection levels were 0.1 to 5% in fresh and processed juice, including pasteurization and HTHP.

Establishment of a sensitive TaqMan-based real-time PCR assay for porcine circovirus type 3 and its application in retrospective quarantine of imported boars to China.

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.