• Journal of Practical Agriculture & Fisheries Research
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• v.5 no.1
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• pp.57-63
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• 2003

The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.339-347
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• 2012

Viral safety is an important prerequisite for clinical preparations of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to several RNA viruses including reovirus (Reo), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus (BPIV) and there have been reports of such viral contaminations. Therefore, viral detection during the CHO cell process is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex reverse transcription (RT)-PCR assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect Reo, BVDV, and BPIV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for Reo, BVDV, and BPIV were selected, and a multiplex RT-PCR was optimized. The sensitivity of the assay for simultaneous amplification of all viral target RNAs was $7.76{\times}10^2\;TCID_{50}/ml$ for Reo, $7.44{\times}10^1\;TCID_{50}/ml$ for BVDV, and $6.75{\times}10^1\;TCID_{50}/ml$ for BPIV. The multiplex RT-PCR was proven to be very specific to Reo, BVDV, and BPIV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral RNA from CHO cells as well as culture supernatants. Therefore, it was concluded that the multiplex RT-PCR assay can be applied to detection of the adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1165-1169
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• 2012

In April 2009, the H1N1 pandemic influenza virus emerged as a novel influenza virus. The aim of this study was to compare the performances of several molecular assays, including conventional reverse transcription polymerase chain reaction (RT-PCR), two real-time reverse transcription (rRT)-PCRs, and two multiplex RTPCRs. A total of 381 clinical specimens were collected from patients (223 men and 158 women), and both the Seeplex RV7 assay and rRT-PCR were ordered on different specimens within one week after collection. The concordance rate for the two methods was 87% (332/381), and the discrepancy rate was 13% (49/381). The positive rates for the molecular assays studied included 93.1% for the multiplex Seeplex RV7 assay, 93.1% for conventional reverse transcription (cRT)-PCR, 89.7% for the multiplex Seeplex Flu ACE Subtyping assay, 82.8% for protocol B rRT-PCR, and 58.6% for protocol A rRT-PCR. Our results showed that the multiplex Seeplex assays and the cRT-PCR yielded higher detection rates than rRT-PCRs for detecting the influenza A (H1N1) virus. Although the multiplex Seeplex assays had the advantage of simultaneous detection of several viruses, they were time-consuming and troublesome. Our results show that, although rRT-PCR had the advantage, the detection rates of the molecular assays varied depending upon the source of the influenza A (H1N1)v virus. Our findings also suggest that rRT-PCR sometimes detected virus in extremely low abundance and thus required validation of analytical performance and clinical correlation.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.210-217
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• 2018

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

• Research in Plant Disease
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• v.24 no.4
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• pp.348-352
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• 2018

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed for the detection of Clover yellow vein virus (ClYVV), Peanut mottle virus (PeMoV), and Tomato spotted wilt virus (TSWV), which were recently reported to infect soybean and azuki bean in Korea. Species-specific primer sets were designed for the detection of each virus, and their specificity and sensitivity were tested using mixed primer sets. From among the designed primer sets, two combinations were selected and further evaluated to estimate the detection limits of uniplex, duplex, and multiplex RT-PCR. The multiplex RT-PCR assay could be a useful tool for the field survey of plant viruses and the rapid detection of ClYVV, PeMoV, and TSWV in leguminous plants.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1164-1169
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• 2008

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus-negative specimens. Furthermore, the assays could detect and subtype up to $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

• The Plant Pathology Journal
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• v.22 no.2
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• pp.168-173
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• 2006

To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the N-terminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RT-PCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor $1{\alpha}(EF1{\alpha})$. This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.

• Research in Plant Disease
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• v.24 no.3
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• pp.233-236
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• 2018

A multiplex RT-PCR (mRT-PCR) assay was developed for the detection of the recently reported viruses, Cherry virus A (CVA), Little cherry virus 1 (LChV-1), and Little cherry virus 2 (LChV-2), in cherry plants in Korea. Eight sets of primers were designed for each virus and their specificity was tested by using various combinations of mixed primer sets. From the designed primer sets, one combination was selected and further evaluated to estimate the optimum temperature and detection limits of the mRT-PCR. A newly developed mRT-PCR assay was also tested using 20 cherry samples collected in the field. This mRT-PCR assay may be a useful tool for field surveys of diseases and the rapid detection of these three viruses in cherry plants.

• Clinical and Experimental Pediatrics
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• v.52 no.12
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• pp.1358-1363
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• 2009

Purpose:The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in children using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. Methods:We obtained nasopharyngeal swabs from 33 children without any underlying disease from July 25 to July 28, 2008. The children were free from the signs of respiratory tract infections at the time of sampling. DNA was extracted from the swabs and subjected to multiplex RT-PCR using a primer set for the detection of pneumococci ($Seeplex^{(R)}$ PneumoBacter ACE Detection Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with either ethidium bromide or screen tape system (Lab901 Scotland, UK). Results:A total of 33 children (male, 15 female, 18) aged between 3.2 and 16.3 (median, 8.2) years were included in this study. The mRT-PCR detected colonized bacteria (Streptococcus pneumoniae, Hemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 30 children (90.9%). Of these, 13 children (39.4%) showed more than 2 bacteria: 12 children were positive for 2 bacteria (S. pneumoniae and H. influenzae) and 1 child was positive for 3 bacteria (S. pneumoniae, H. influenzae, and C. pneumoniae). Conclusion:mRT-PCR was found to be a sensitive tool for the detection of asymptomatic nasopharyngeal carriages. Clinical significances of the bacteria detected by mRT-PCR will have to be evaluated in the future.

• Pediatric Infection and Vaccine
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• v.25 no.2
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• pp.91-100
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• 2018

Purpose: This study investigated the factors affecting the use of empirical antibiotics in febrile infants from 1 month to less than 3 months. Methods: We retrospectively reviewed the medical records of hospitalized previously healthy infants with fever in Pusan National University Children's Hospital from January 2010 to December 2016. Clinical features, laboratory findings and antibiotic therapy were analyzed. Respiratory viruses were identified by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and were reported after 1-3 days. Enterovirus were identified by real time polymerase chain reaction (PCR) and were reported in several hours. Results: The 129 of 366 subjects used empirical antibiotics and 237 patients didn't used empirical antibiotics. Empirical antibiotics were used more frequently when the fever was longer before admission, respiratory symptoms and ill being appearances were present and C-reactive protein was elevated. The rate of readmission was low in the group not used empirical antibiotics. Most of the patients detected by enterovirus PCR in cerebrospinal fluid didn't used empirical antibiotics. The results of respiratory virus multiplex RT-PCR showed no difference in the use of empirical antibiotics. Conclusions: In our study, empirical antibiotic prescriptions were affected not respiratory virus multiplex RT-PCR but enterovirus PCR. If multiplex RT-PCR were reported more rapid turn around time, it will affect antibiotic use.