• Parasites, Hosts and Diseases
• /
• v.49 no.3
• /
• pp.281-284
• /
• 2011

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

• Parasites, Hosts and Diseases
• /
• v.60 no.4
• /
• pp.295-299
• /
• 2022

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

• Tuberculosis and Respiratory Diseases
• /
• v.68 no.6
• /
• pp.345-349
• /
• 2010

Background: The polymerase chain reaction (PCR) test is important for the confirmatory diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis. The aim of this study was to analyze the yield of repeated PCR testing in patients with confirmed pulmonary TB. Methods: The medical records of 130 patients, who had more than two consecutive PCR tests and a M. tuberculosis-positive sputum culture from August, 2006 to December, 2007, were retrospectively reviewed for the purposes of this study. A positive TB-PCR test was defined as at least one positive test result. Results: The cumulative positive PCR test rate was 80% (104/130), with gradually increasing rates of positive findings upon the first, second and third TB-PCR tests with 52.3%, 68.5% and 75.4%, respectively. However, further testing did not increase the positive rate further. Conclusion: Repeated PCR testing at least three times for M. tuberculosis is helpful for diagnosis of pulmonary TB.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.8
• /
• pp.1113-1117
• /
• 2012

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Korean Journal of Clinical Laboratory Science
• /
• v.47 no.4
• /
• pp.313-317
• /
• 2015

Due to the increase in incidence of infection of Mycobacterium tuberculosis complex (MTC), it is imperative that a rapid diagnosis accompanies the handling of MTC. This is due to the three to eight weeks it takes to culture Mycobacteria, and the lack of sensitivity of microscopic examination of AFB. Recently, nested PCR has been used to detect and diagnose mycobacteria. It is especially useful in complementing diagnosis by histological extra pulmonary. After culturing all the specimens and practicing the nested PCR, we did comparison analysis between nested PCR and culture. There were 76 specimens, 31 of which were positive. Of the 31 positive specimens in culturing, only 22 were positive in nested PCR. Of the 45 negative specimens, 36 were negative in nested PCR. As a result, Sensitivity was 71% and specificity was 80%. Furthermore, the positive predictive value was 71% and negative predictive value was 80%. These results indicate that nested PCR based techniques are sensitive, specific, and rapid methods for the detection of MTC.

• Research in Plant Disease
• /
• v.11 no.1
• /
• pp.48-55
• /
• 2005

Cylindrocarpon destructans is a soil-borne plant pathogenic fungus causing root rot on ginseng and trees. Rapid and exact detection of this pathogen was practiced on ginseng seedlings by nested PCR using speciesspecific primer set. The second round of PCR amplification by Dest 1 and Dest 4 primer set formed 400 bp of species-specific fragment of C. destructans from the product of first round of amplification by ITS 1 and ITS 4 primer set. In the PCR sensitivity test based on DNA density, nested PCR detected to the limit of one fg and it meant the nested PCR could detect up to a few spores of C. destructans. Also, nested PCR made it possible to detect the pathogen from ginseng seedlings infected by replantation on artificial infested soil. Our nested PCR results using species-specific primer set could be utilized for diagnosis of root rot disease in ginseng cultivation.

• Journal of Genetic Medicine
• /
• v.12 no.2
• /
• pp.72-78
• /
• 2015

Recently, noninvasive prenatal test (NIPT) has been adopted as a primary screening tool for fetal chromosomal aneuploidy. The principle of NIPT lies in isolating the fetal fraction of cell-free DNA in maternal plasma and analyzing it with bioinformatic tools to measure the amount of gene from the target chromosome, such as chromosomes 21, 18, and 13. NIPT will contribute to decreasing the need for unnecessary invasive procedures, including amniocentesis and chorionic villi sampling, for confirming fetal aneuploidy because of its higher positive predictive value than that of the conventional prenatal screening method. However, its greater cost than that of the current antenatal screening protocol may be an obstacle to the adoption of this innovative technique in clinical practice. Digital polymerase chain reaction (dPCR) is a novel approach for detecting and quantifying nucleic acid. dPCR provides real-time diagnostic advantages with higher sensitivity, accuracy, and absolute quantification than conventional quantitative PCR. Since the groundbreaking discovery that fetal cell-free nucleic acid exists in maternal plasma was reported, dPCR has been used for the quantification of fetal DNA and for screening for fetal aneuploidy. It has been suggested that dPCR will decrease the cost by targeting specific sequences in the target chromosome, and dPCR-based noninvasive testing will facilitate progress toward the implementation of a noninvasive approach for screening for trisomy 21, 18, and 13. In this review, we highlight the principle of dPCR and discuss its future implications in clinical practice.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.58 no.2
• /
• pp.142-148
• /
• 2013

Bacterial diseases in soybean are bacterial pustule by Xanthomonas axonopodis pv. glycines, wildfire by Pseudomonas syringae pv. tabaci, bacterial blight by Pseudomonas savastanoi pv. glycines and bacterial brown spot by Pseudomonas syringae pv. syringae in Korea. It is difficult to identify each disease by early symptoms in fields, because the initial symptoms of these diseases are very similar to each other. In this study, we developed multiplex PCR detection method for rapid and accurate diagnosis of bacterial diseases. The glycinecin A of X. axonopodis pv. glycines, the tabtoxin of P. syringae pv. tabaci, the coronatine of P. savastanoi pv. glycines and the syringopeptin of P. syringae pv. syringae have been reported previously. These bacteriocin or phytotoxin producing genes were targeted to design the specific diagnostic primers. The primer pairs for diagnosis of each bacterial diseases were selected without nonspecific reactions. The studies on simultaneous diagnosis method were also conducted with primarily selected 21 primers. As a result, we selected PCR primer sets for multiplex PCR. Sizes of the amplified PCR products using the multiplex PCR primer set consist of 280, 355, 563 and 815 bp, respectively. This multiplex PCR method provides a efficient, sensitive and rapid tool for the diagnosis of the bacterial diseases in soybean.

• Tuberculosis and Respiratory Diseases
• /
• v.75 no.4
• /
• pp.150-156
• /
• 2013

Background: Thoracoscopic pleural biopsy is often required for rapid and confirmative diagnosis in patients with suspected pleural tuberculosis (PL-TB). However, this method is more invasive and costly than its alternatives. Therefore, we evaluated the clinical utility of the chest computed tomography (CT)-based bronchial aspirate (BA) TB-polymerase chain reaction (PCR) test in such patients. Methods: Bronchoscopic evaluation was performed in 54 patients with presumptive PL-TB through diagnostic thoracentesis but without a positive result of sputum acid-fast bacilli (AFB) smear, pleural fluid AFB smear, or pleural fluid TB-PCR test. Diagnostic yields of BA were evaluated according to the characteristics of parenchymal lesions on chest CT. Results: Chest radiograph and CT revealed parenchymal lesions in 25 (46%) and 40 (74%) of 54 patients, respectively. In cases with an absence of parenchymal lesions on chest CT, the bronchoscopic approach had no diagnostic benefit. BA TB-PCR test was positive in 21 out of 22 (95%) patients with early-positive results. Among BA results from 20 (37%) patients with patchy consolidative CT findings, eight (40%) were AFB smear-positive, 18 (90%) were TB-PCR-positive, and 19 (95%) were culture-positive. Conclusion: The BA TB-PCR test seems to be a satisfactory diagnostic modality in patients with suspected PL-TB and patchy consolidative CT findings. For rapid and confirmative diagnosis in these patients, the bronchoscopic approach with TB-PCR may be preferable to the thoracoscopy.

• Genomics & Informatics
• /
• v.21 no.1
• /
• pp.13.1-13.8
• /
• 2023

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.