• Parasites, Hosts and Diseases
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• 제47권3호
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• pp.293-297
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• 2009

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from $10^3$ to $10^4$ oocysts, and the nested PCR method was able to detect $10^0$ to $10^2$ oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.

• Food Science and Biotechnology
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• 제15권4호
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• pp.625-630
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• 2006

Polymerase chain reaction (PCR) method was developed for the specific detection of insect-resistant rice containing cry1Ac gene derived from Bacillus thuringiensis (Bt). Primers were designed from the 35S promoter, NOS terminator, cry1Ac gene, and sucrose phosphate synthase (SPS) for general screening of Bt rice. By sequencing the PCR products from the two putative kinds of Bt rice, we designed a specific primer from the junction region between the cry1Ac gene and the NOS terminator that had been inserted into Bt rice. The construct-specific primer was employed to amplify a 147 bp product in the two lines of Bt rice. No amplified products were observed from the other Bt crops with various Bt genes introduced. In qualitative PCR analysis, the limit of detection was 0.005 ng from genomic DNA of Bt rice. In addition, PCR analysis was performed on 64 kinds of rice presently available in the Korean market, and no Bt rice was detected. This method presented in this paper can be used as a highly sensitive and specific detection method of Bt rice.

• IMMUNE NETWORK
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• 제24권2호
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• pp.12.1-12.17
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• 2024

Exosomes are double phospholipid membrane vesicles that are synthesized and secreted by a variety of cells, including T cells, B cells, dendritic cells, immune cells, are extracellular vesicles. Recent studies have revealed that exosomes can play a significant role in under both physiological and pathological conditions. They have been implicated in regulation of inflammatory responses, immune response, angiogenesis, tissue repair, and antioxidant activities, particularly in modulating immunity in autoimmune diseases (AIDs). Moreover, variations in the expression of exosome-related substances, such as miRNA and proteins, may not only offer valuable perspectives for the early warning, and prognostic assessment of various AIDs, but may also serve as novel markers for disease diagnosis. This article examines the impact of exosomes on the development of AIDs and explores their potential for therapeutic application.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1672-1683
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• 2021

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla_CARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla_CARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg²⁺, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63℃ for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10^-4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1146-1153
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• 2015

This study was conducted to assess the relationship between occurrence of gastric cancer and peptic ulcer, and the presence of H. pylori cagA gene and anti-CagA IgG, and to estimate the value of these antibodies in detecting infection by cagA gene-positive H. pylori strains in Saudi patients. The study included 180 patients who were subjected to upper gastrointestinal endoscopy in Taif province and Western region of Saudi Arabia (60 gastric cancer, 60 peptic ulcer, and 60 with non-ulcer dyspepsia). Gastric biopsy specimens were obtained and tested for H. pylori infection by rapid urease test and culture. PCR was performed on the isolated strains and biopsy specimens for detection of the cagA gene. Blood samples were collected and tested for CagA IgG by ELISA. H. pylori infection was detected among 72.8% of patients. The cagA gene and anti-CagA IgG were found in 63.4% and 61.8% of H. pylori-infected patients, respectively. They were significantly (p < 0.01) higher in patients with gastric cancer and peptic ulcer compared with those with non-ulcer dyspepsia. Detection of the CagA IgG was 91.6% sensitive, 89.6% specific, and 90.8% accurate compared with detection of the cagA gene. Its positive and negative predictive values were 93.8% and 86%, respectively. The study showed a significant association between the presence of the cagA gene and gastric cancer and peptic ulcer disease, and between anti-CagA IgG and the cagA gene in Saudi patients. However, a further larger study is required to confirm this finding.

• 대한미생물학회지
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• 제34권6호
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• pp.513-518
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• 1999

A total of 113 Vibrio sp. strains were examined for plasmid content which were subjected to digestion with restriction enzymes. About the 55% Vibrio spp. have the plasmid more than one. Most of these plasmid various derivatives ranged from $2.4\;kb{\sim}23\;kb$, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between $70\;kb{\sim}100\;kb$). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH and CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 9 strains of V. parahaemolyticus and 1 strain of V. alginolyticus from clinical isolates and 1 strains of V. mimicus from environmental isolates. In the experiments of tdh gene detection, in all, 3 strains of V. parahaemolyticus from clinical isolates and 2 strains from environmental isolates could be successfully amplified in 400 bp by PCR. The PCR results were consistent with DNA hybridization tests. In the experiments of CT assay, in all, 3 strains of V. cholerae from clinical isolate and 1 strain of V. cholerae from environmental isolates were observed CT-producing. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.

• 한국동물위생학회지
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• 제22권3호
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• pp.239-245
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• 1999

The reverse transcriptase polymerase chain reaction (RT-PCR) was used for the detection of bovine coronavirus (BCV) in fecal samples by using reverse transcriptase and two primers which flanked M gene sequence of 407bp. RT-PCR detected bovine coronavirus specifically, but did not detect mouse hepatitis virus (MHV), transmissible gastroenteritis virus (TGEV), and bovine rotavirus (BRV). The M gene sequences of MHV are homologus to that of BCV, but minor differences exist in the primer regions, preventing annealing of the primers. Detection of BCV using RT-PCR was compared with ELISA and the agreement of BCV detection by RT-PCR and ELISA was 95.3%. RNA detection in positive clinical specimens was significantly better by PCR than immunological detection of BCV by ELISA.

• Journal of Veterinary Science
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• 제24권3호
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• pp.40.1-40.6
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• 2023

Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples.

• Pediatric Infection and Vaccine
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• 제3권2호
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• pp.133-138
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• 1996

Purpose : In the treatment of MRSA infection, rapid detection of MRSA is extremely important. The mecA gene codes the new drug resistant polypeptides called PBP2' which mediates the clinically relevant resistance to all beta-lactam antibiotics. The identical mecA gene has been found in coagulase-negative staphylococcus with the methicillin-resistant phenotype. On the other hand, the femA gene was absent from coagulase negative staphylococcus strains with the methicillin resistant phenotype. This study is aimed at early detection and definite diagnosis of MRSA. Methods : A total of 24 MRSA strains were studied. All strains were tested for antimicrobial susceptibility and purified DNA. We amplified both mecA and femA genes by PCR in 24 strains. Results : In MRSA all the 16 strains (100%) carried femA gene and 11 strains (68.7%) carried mecA gene. In contrast, in methicillin sensitive staphylococcus all the 8 strains (100%) carried femA and only 3 strains (37.5%) were detected mecA. Conclusions : As results, there are difference in the phenotype and genotype of methicillin resistance by PCR of mecA and femA. Such disparities between methicillin resistance and the presence of mecA gene suggest the presence of control gene of the mecA.

• 대한의생명과학회지
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• 제11권4호
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• pp.447-453
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• 2005

A total of 108 strains of MRSA (Methicillin-resistant Staphylococcus aureus) clinical isolates was collected from $121^{st}$ general hospital (U.S. military hospital), Korean healthcare facility from January to March in 2005 and Wonju Christian hospital in 1999. Antimicrobial susceptibility test by Vitek System and MIC test using oxacillin and cephalothin stripes by E-test were executed. PCR based detection of mecA gene was performed on the all of MRSA clinical isolates, too. MRSA clinical isolates were characterized with antimicrobial resistance patterns, PCR based detection of mecA gene and validation of the multiplex PCR strategy of SCCmec among clinical isolates.