• Preventive Nutrition and Food Science
• /
• v.17 no.4
• /
• pp.274-279
• /
• 2012

For the quantitative analysis of genetically modified (GM) maize in processed foods, primer sets and probes based on the 35S promoter (p35S), nopaline synthase terminator (tNOS), p35S-hsp70 intron, and zSSIIb gene encoding starch synthase II for intrinsic control were designed. Polymerase chain reaction (PCR) products (80~101 bp) were specifically amplified and the primer sets targeting the smaller regions (80 or 81 bp) were more sensitive than those targeting the larger regions (94 or 101 bp). Particularly, the primer set 35F1-R1 for p35S targeting 81 bp of sequence was even more sensitive than that targeting 101 bp of sequence by a 3-log scale. The target DNA fragments were also specifically amplified from all GM labeled food samples except for one item we tested when 35F1-R1 primer set was applied. A reference plasmid pGMmaize (3 kb) including the smaller PCR products for p35S, tNOS, p35S-hsp70 intron, and the zSSIIb gene was constructed for real-time PCR (RT-PCR). The linearity of standard curves was confirmed by using diluents ranging from $2{\times}10^1{\sim}10^5$ copies of pGMmaize and the $R^2$ values ranged from 0.999~1.000. In the RT-PCR, the detection limit using the novel primer/probe sets was 5 pg of genomic DNA from MON810 line indicating that the primer sets targeting the smaller regions (80 or 81 bp) could be used for highly sensitive detection of foreign DNA fragments from GM maize in processed foods.

• Biomedical Science Letters
• /
• v.22 no.4
• /
• pp.215-219
• /
• 2016

The problems of tuberculosis and its drug resistance are very severe. Therefore, rapid and accurate drug susceptibility assay is required. Recently, there has been an increased understanding of the genetic mechanism of Mycobacterium tuberculosis (MTB) drug resistance as well as advancement of molecular technologies. While many gene mutations correlate well with drug resistance, many genes do not show a strong correlation with drug resistance. For this reason, the current study assessed the utility of rpoB mRNA as a target to detect live mycobacteria. In this study, RT-PCR targeting of rpoB mRNA in BCG treated with rifampin was performed. Conventional RT-PCR and real-time PCR targeting rpoB mRNA as well as 85B mRNA was performed to determine whether these two methods could distinguish between viable and non-viable MTB. The levels of rpoB and 85B mRNA detected by RT- PCR were compared in parallel with colony forming unit counts of BCG that were treated with rifampin for different periods of time. The data suggests that that even though both mRNA levels of rpoB and 85B decreased gradually when rifampin-treatment increased, the rpoB mRNA seemed to represent live bacteria better than 85B mRNA. This study clearly indicates that RT-PCR is a good method to monitor viable cell counts in the liquid culture treated with the anti-tuberculosis drug.

• Biomedical Science Letters
• /
• v.13 no.1
• /
• pp.33-38
• /
• 2007

The aim of this work was to validate a rapid and an accurate method for detecting Mycobacterium leprae in clinical specimens using nested PCR targeting M. leprae-specific repetitive element (RLEP) sequence. The primers were derived from the RLEP sequence which yield a 272 bp outer product and a 230 bp inner product. The specificity and the sensitivity of the nested PCR were compared with those of single PCR for detecting M. leprae using DNAs isolated from reference strain and various species of Mycobacterium. The results showed that the sensitivity of the nested PCR was about 100 to 1,000 times higher than that of the single PCR and also showed that both the single and the nested PCR were highly specific to M. leprae. Subsequently, the usefulness of the single and nested PCR was evaluated with clinical samples isolated from leprosy patients. The number of positive detections by the single and the nested PCR with a total of 20 specimens from leprosy patients were 9 (45%) and 20 (100%), respectively. The results clearly showed that nested PCR has highest sensitivity in detecting M. leprae from clinical specimens. Therefore, nested primers targeting RLEP sequence developed in this study seems to be useful to detect the presence of M. leprae.

• Biomedical Science Letters
• /
• v.16 no.2
• /
• pp.127-131
• /
• 2010

Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens.

• Parasites, Hosts and Diseases
• /
• v.54 no.3
• /
• pp.329-334
• /
• 2016

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.

• Journal of Microbiology
• /
• v.40 no.4
• /
• pp.274-281
• /
• 2002

The competitive quantitative PCR method targeting pcbC gene was developed for monitoring 4-chlorobiphenyl(4CB)-degrading bacteria, Pseudomonas sp. strain DJ-12, in soil microcosms. The method involves extraction of DNA from soil contaminated with 4CB, PCR amplification of a pcbC gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the elec-trophoresed PCR product by densitometry. To test the adequacy of the method, Pseudomonas sp. strain DJ-12 was introduced into both contaminated and non-contaminated soil microcosms amended with 4CB. Pseudomonas sp. strain DJ-12 was monitored and quantified by a competitive quantitative PCR in comparison with 4CB degradation and the result was compared to those obtained by using the conventional cultivation method. We successfully detected and monitored 4CB-degrading bacteria in each microcosm and found a significant linear relationship between the number of 4CB-degrading bacteria and the capacity for 4CB biodegradation. The results of DNA spiking and cell-spreading experiments suggest that this competitive quantitative PCR method targeting the pcbC gene for monitoring 4CB- degrading bacteria appears to be rapid, sensitive and more suitable than the microbiological approach in estimating the capacity of 4CB biodegradation in environmental samples.

• The Plant Pathology Journal
• /
• v.39 no.4
• /
• pp.309-318
• /
• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.47 no.5
• /
• pp.556-561
• /
• 2014

We applied a combination of most probable number-polymerase chain reaction (MPN-PCR) methods using a PCR procedure targeting the H-NS (VP1133) gene to detect Vibrio parahaemolyticus presence and density in seawater as well as within short-necked clam Ruditapes philippinarum tissues collected from Gomso Bay, Korea. In 30 seawater samples, V. parahaemolyticus levels ranged from less than 1.8 to $1.1{\times}10^3MPN/100mL$, and samples from August showed higher than those from other months. Furthermore, the levels of V. parahaemolyticus in six short-necked clam samples ranged from $7.8{\times}10^2$ to $2.1{\times}10^3MPN/100g$, approximately 2.5 times higher than in seawater samples from the corresponding month. Our results provide data on V. parahaemolyticus contamination in seawater and short-necked clam tissues, and help to improve quantitative methods of assessing V. parahaemolytcius levels.

• Genomics & Informatics
• /
• v.19 no.3
• /
• pp.30.1-30.8
• /
• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2005.05a
• /
• pp.206.4-206
• /
• 2005