• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• Tuberculosis and Respiratory Diseases
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• v.70 no.4
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• pp.315-322
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• 2011

Background: A variety of diagnostic modalities for lung cancer have been developed. To achieve efficient and early detection of lung cancer, we tried to measure the expression rates of the melanoma associated gene (MAGE) and synovial sarcoma on X chromosome (SSX) genes. Methods: We designed primers for the SSX gene. In addition to the pre-developed MAGE A primer, using an SSX gene primer was attempted to increase the detection rate. We obtained cancer tissues and cancer-free lung tissues from resected lung, sputum from lung cancer patients who had not undergone surgery, and sputum from healthy people and patients with benign intrathoracic diseases. Results: The sensitivity of the MAGE or SSX gene RT-PCR to identifying cancer tissue of the 69 lung cancer patients was 95.2% for squamous cell carcinoma (scc), 87.0% for adenocarcinoma, and 100% for small cell carcinoma. The mean sensitivity value was 94.2% (p=0.001). For adenocarcinoma, the additional use of the SSX gene resulted in a higher expression rate than MAGE alone (87% vs. 69.6%). The expression rate for the cancer-free lung tissue was 14.3% in scc, 17.4% in adenocarcinoma, and 25.0% in small cell carcinoma. In the induced sputum of 49 lung cancer patients who had not undergone surgery, the expression rate for one of the two genes was 65.5%. The expression rate for the sputum of healthy people and benign intrathoracic diseases by MAGE or SSX gene reverse transcription polymerase chain reaction (RT-PCR) was 3.8% and 17.7%. Conclusion: Detecting lung cancer using the expression of MAGE and SSX genes in lung cancer tissue has high sensitivity.

• The Plant Pathology Journal
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• v.36 no.5
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• pp.459-467
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• 2020

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrI-B subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64℃ for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/μl, namely approximately 53 copies/μl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

• Journal of Veterinary Science
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• v.23 no.4
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• pp.51.1-51.10
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• 2022

Background: Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives: The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods: LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results: Our results showed that the LMTIA assay could detect the 10⁴ dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions: The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

• KSBB Journal
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• v.20 no.4
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• pp.323-327
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• 2005

RT-PCR is an useful method to investigate the expression of target gene as detection tools. Although RT-PCR is the powerful detection method for tissues, it was difficult to amplify the target gene product using the single cell. To clarify the expression level of the genes related to Parkinson's disease (PD), I performed the laser dissection of single cell from Substantia nigra. I examined the mRNA expression level in the dopaminergic neuron isolated from the PD patients by the single cell RT-PCR method. It is known that tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) are involved in biosynthesis of the catecholamine such as dopamine. Little has been known about the gene expression features of these enzymes in single dopaminergic neuron. I could detect the specific gene products in single cell level. The different expression was observed in PD-related gene products from the single neuron of PD patients. Interestingly, TH gene expression was significantly decreased with comparing the ratio of decrease in other PD-related genes. Hence, I represented data that indicate the RT-PCR method described in this report is an effective method in detecting a specific single-cell mRNA level related with diseases.

• Animal Bioscience
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• v.36 no.4
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• pp.654-670
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• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

• Clinical and Experimental Pediatrics
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• v.53 no.12
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• pp.1022-1025
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• 2010

Thanatophoric dysplasia (TD) is a short-limb neonatal dwarfism syndrome that is usually lethal in the perinatal period. It is characterized by shortening of the limbs, severely small thorax, large head with a prominent forehead, macrocephaly, curved femur, and flattened vertebral bodies. These malformations result from the mutation in fibroblast growth factor receptor 3 (FGFR-3) gene which is located on the short arm of chromosome 4. A definite diagnosis should be established by molecular genetic analysis to find out the abnormal mutations in the $FGFR3$ gene. We confirmed by detection of a R248C mutation in the $FGFR3$ gene in DNA analysis.

• Journal of Genetic Medicine
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• v.3 no.1
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• pp.15-19
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• 1999

We have investigated the genetic variation in the human apo B mRNA editing protein (apobec-1) gene. Exon 3 of the apobec-1 gene was amplified by polymerase chain reaction. After detection of an additional band by single strand conformational polymorphism (SSCP) analysis, sequencing of the SSCP-shift sample revealed a single-base mutation. The mutation was a CGG transversion at codon 80 resulting in a lleRMet substitution. This substitution was confirmed by restriction fragment length polymorphism analysis since a Pvull site is abolished by the substitution. Population and family studies confirmed that the inheritance of the genotypes for apobec-1 gene polymorphism is controlled by two codominant alleles (P1 and P2). A significant difference in plasma triglyceride was detected among the different apobec-1 genotypes in the CAD patients (P<0.05). Our study could provide the basis for elucidating the interaction between genetic variation of the apobec-1 gene and disorders related to lipid metabolism.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.109-118
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• 1996

The Salmonella rfb gene encoding for the biosynthesis of the oligosaccharide-repeating units of the O-antigenic determinants was cloned and sequenced. A set of nucleotide primers(a forward and reverse) was selected to target a defined region of the guanosine diphospho-mannose(GDP-Man) pyrophosphorylase synthase gene : rfbM of Salmonella C serogroup. The primer set was used to develop a PCR-based rapid and specific detection system for Salmonella C1 serogroup. Amplification bands of predicted size(1,422bp) were generated from 11 different Salmonella C1 isolates. The bands were verified to be specific for the C1 serogroup by Southern blot analysis using reference homologous DNA specificity was further confirmed by the lack of reactivity with heterologous DNA derived from non-salmonella members of the family enterobacteriaeceae. A specificity of 100% was deduced along with a very high sensitivity shown by a detection limit of 1fg of a purified DNA template. The isolated DNA sequence was found to be 99.8% homologous to S montevideo but the related primers amplified with the predicted band sizes with all the Salmonella C1 serogroups tested. It is concluded that the PCR protocol based on the rfbM gene from S cholerasuis is optimal fast and specific for the detection of Salmonella C1 serogroup and also the corresponding probe is suitable for rapid detection of all Salmonella C1 serogroup DNA tested. This technology should facilitate the identification of contaminated pig products and for any other products contaminated with the Salmonalla C1 serogroup. The immediate impact of this developed method will be in the area of food safety of pig products with the potential prospect for adaptation to other food inspection technologies.

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.6
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• pp.1016-1024
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• 2000

The study was conducted to develop a rapid method for the detection of Listeria monocytogenes in foods via polymerase chain reaction (PCR) technique using hemolysin gene (hlyA) primers. Specificity and sensitivity of PCR, optimal conditions for PCR and application of hlyA gene primers for the detection of L. monocytogenes from milk and beef were investigeted. Each of the 20 L. monocytogenes strains gave a single 713 bp band, but other Listeria sup. and other bacteria did not show any bands. As few as 1 pg of L. monocytogenes DNA or 2.4$\times$10$^4$L. monocytogenes cells could be detected with hlyA gene primers. PCR product was most improved at 20~30 cycle in terms of removal of tailing and sensitivity. Also, the sensitivity was significantly improved by the further 10~15 cycle after 20 cycle PCR amplication. Milk (10 mL) and beef (10 g) samples were inoculated with L. monocytogenes at the concentrations ranging from 0 to 10$^{7}$ CFU/mL or g to determine the best sensitivity of PCR for the rapid detection of L. monocytogenes. PCR assay could detect 2 cells in milk with repeating PCR amplication and 2.6$\times$10$^2$cells in beef sample after 24 hr enrichment growth at 35$^{\circ}C$ in LEB.