• 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.

• Korean Journal Plant Pathology
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• v.12 no.3
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• pp.358-362
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• 1996

감자 잎말림 바이러스를 검정하기 위하여 ELISA 및 전자현미경에 의해 바이러스 감염이 확인된 기내 배양중인 감장의 줄기로부터 RT-PCR 분석을 수행하였다. 분리된 총 RNA들로부터 바이러스 cDNA를 합성하고 감자 잎말림 바이러스 외피단백질의 일부인 465bp를 특이하게 증폭하도록 고안한 두 primer를 사용하여 PCR 반응을 하였다. 증폭된 465pb의 DNA 절편의 염기서열을 분석한 결과 역시 감자 잎말림 바이러스임을 확인하였다. 바이러스 검정에 있어서 EL-ISA 방법과 RT-PCR 방법간의 민감도를 조사한 결과 RT-PCR 방법간의 민감도를 조사한 결과 RT-PCR 방법이 ELISA 방법보다 감자 잎말림 바이러스검정에 있어서보다 정확한 방법인 것으로 사료된다.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.270-274
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• 2013

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

• Research in Plant Disease
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• v.17 no.1
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• pp.44-51
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• 2011

The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv), Clavibacter michiganensis subsp. michiganensis (Cmm), Erwinia carotovora subsp. carotovora (Ecc), Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV) in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R) of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.44 no.3
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• pp.253-255
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• 1999

Reverse transcription and polymerase chain reaction (RT-PCR) assay was used to detect SMV strains. A pair of oligonucleotide primers were designed to include the cylindrical inclusion (CI) coding region between 4,176 to 5,560 nt. Amplification from the total RNA extracted from infected plants with SMV yielded a 1,385 bp DNA fragment. RT-PCR was shown to be $10^3$ times more sensitive than the ELISA assay and it could detect a virus in $10^{-6}$ dilution. Restriction enzyme analysis of RT- PCR products using EcoR I showed that SMV isolates were classified into six groups according to the patterns of restriction fragments.

• Journal of Life Science
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• v.29 no.6
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• pp.653-661
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• 2019

The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.

• The Plant Pathology Journal
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• v.26 no.1
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• pp.25-31
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• 2010

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

• 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.

• Research in Plant Disease
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• v.17 no.1
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• pp.52-58
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• 2011

The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc) and Lettuce Mosaic Virus (LMV). Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage), two pairs of specific primer (LMV-F/R, Xcc-F/R) were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties), lettuce (50 varieties), radish (20 varieties), chinese cabbage (20 varieties) and cabbage (20 varieties), LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.

• Biomedical Science Letters
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• v.22 no.3
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• pp.83-97
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• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.