Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

Enhanced detection and serotyping of foot-and-mouth disease virus serotype O, A, and Asia1 using a novel multiplex real-time RT-PCR.

Rapid and accurate detection and serotyping of foot-and-mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription-polymerase chain reaction (RT-PCR)/sequencing (VP1 RT-PCR/sequencing) and antigen detection enzyme-linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real-time RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real-time RT-PCR using these primers and probes demonstrated serotype-specific detection with enhanced sensitivity compared to VP1 RT-PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real-time RT-PCR and previous VP1 RT-PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real-time RT-PCR and VP1 RT-PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real-time RT-PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real-time RT-PCR and VP1 RT-PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real-time RT-PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.

Development and application of DNA markers to detect adulteration with Scopolia japonica in the medicinal herb Atractylodes lancea.

Atractylodes lancea rhizomes are commonly consumed in east Asia as traditional medical herbs. However, in Korea, because of their morphological similarity, A. lancea rhizomes can be contaminated with those of Scopolia japonica imported from China. To detect adulteration with S. japonica in the complex products of A. lancea, we developed two PCR-based DNA markers, multiplex PCR and quantitative real-time PCR. The sensitivity of the multiplex PCR primer combinations and real-time PCR was confirmed with a series of DNA concentrations (0.01-10 ng/µL). The specificity of the developed PCR assays was confirmed with 14 other species. In addition, 14 commercial A. lancea medicinal herbs and 20 blind samples were tested with the developed PCR assays to demonstrate the reliability. Taken together, the developed multiplex and real-time PCR-based target-specific primer sets may be useful for detecting the target species and have the potential to contribute to food safety and consumer health care. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-01008-5.

Development of molecular markers to distinguish between morphologically similar edible plants and poisonous plants using a real-time PCR assay.

BACKGROUND: As a result of similar appearances between edible and poisonous plants, 42 patients have ingested poisonous plants from 2013 to 2017 in Korea. We have developed species-specific primer sets of three of edible and poisonous plants sets (Ligularia fischeri & Caltha palustris, Artemisia annua & Ambrosia artemisiifolia and Hemerocallis fulva & Veratrum maackii) for distinguishing both plants using a real-time polymerase chain reaction assay. RESULTS: The efficiencies of the developed primer sets ranged from 87.8% to 102.0%. The developed primer sets have significant correlation coefficient values between the Ct values and the log DNA concentration for their target species (r2 > 0.99). The cut-off lines as the crossing point values of the limit of quantitation of the target species were determined, and all non-target species were amplified later than the cut-off cycles. Then, the effectiveness of the developed primer sets was evaluated using commercial food products and digested samples with simulated gastric juice. CONCLUSION: All of the developed species-specific primer sets were able to detect target DNA successfully in commercial food products and the digested samples. Therefore, the developed species-specific primer sets in the present study would be useful tools for distinguishing between poisonous plants and edible plants. © 2020 Society of Chemical Industry.

Development and Validation of a Real-Time PCR Based Assay to Detect Adulteration with Corn in Commercial Turmeric Powder Products.

Turmeric, or Curcuma longa, is commonly consumed in the South East Asian countries as a medical product and as food due to its therapeutic properties. However, with increasing demand for turmeric powder, adulterated turmeric powders mixed with other cheap starch powders, such as from corn or cassava, are being distributed by food suppliers for economic benefit. Here, we developed molecular markers using quantitative real-time PCR to identify adulteration in commercial turmeric powder products. Chloroplast genes, such as matK, atpF, and ycf2, were used to design species-specific primers for C. longa and Zea mays. Of the six primer pairs designed and tested, the correlation coefficients (R2) were higher than 0.99 and slopes were -3.136 to -3.498. The efficiency of the primers was between 93.14 and 108.4%. The specificity of the primers was confirmed with ten other species, which could be intentionally added to C. longa powders or used as ingredients in complex turmeric foods. In total, 20 blind samples and 10 commercial C. longa food products were tested with the designed primer sets to demonstrate the effectiveness of this approach to detect the addition of Z. mays products in turmeric powders. Taken together, the real-time PCR assay developed here has the potential to contribute to food safety and the protection of consumer's rights.