• Asian-Australasian Journal of Animal Sciences
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• v.33 no.10
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• pp.1683-1690
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• 2020

Objective: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. Methods: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. Results: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). Conclusion: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

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

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.467-472
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• 2018

Objective: Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Results: The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). Conclusion: ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

• Journal of Dairy Science and Biotechnology
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• v.39 no.2
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• pp.51-62
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• 2021

Escherichia coli are the predominant facultative bacteria found in the gastrointestinal tract of animals and humans. Some strains of E. coli that acquire virulence factors and cause foodborne and waterborne diseases in humans are called pathogenic E. coli and can be divided into five pathotypes according to the virulence mechanism: EAEC, EHEC, EIEC, EPEC, and ETEC. Although selective media have been developed to detect E. coli, distinguishing pathogenic strains from non-pathogenic ones is difficult because of their similar biochemical properties. Therefore, it is very important to find a new and effective diagnostic method to identify pathogenic E. coli. With recent advances in molecular biology and whole genome sequencing, the use of polymerase chain reaction (PCR) is increasing rapidly. In this review paper, we provide an overview of pathogenic E. coli and present a review on PCR detection methods that can be used to diagnose pathogenic E. coli. In addition, the possibility of real-time PCR incorporating IAC is introduced. Consequently, this review paper will contribute to solving the current challenges related to the detection of pathogenic E. coli.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.3669-3673
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• 2012

A role for the xenotropic murine leukemia virus (XMRV) in prostate cancer development has been postulated. To answer questions regarding the prevalence of XMRV in Iranian patients with prostate cancer and its association with the RNASEL R462Q polymorphism, we here investigated a series of cases in Kerman, in the Southeast of Iran, and sought to verify the association with the R462Q using Real Time PCR Method. Prostate tissue specimens of 200 patients with prostate cancer were genotyped for R462Q by real time polymerase chain reaction allelic discrimination and were screened for XMRV proviral DNA by real time polymerase chain reaction specific for the envelope gene. Of 200 patients in this study 8 (4%) cases were positive for XMRV, the QQ allele being the most frequenct regarding the R426Q polymorphism while in negative patients it was the RQ allele. There was significant correlation between high pathological scores and XMRV positive samples. No significant relationship was found between age groups and XMRV results. XMRV was only found in patients with QQ and RQ alleles, not RR. XMRV is detectable in tumor prostate tissue from some patients with prostate cancer, independent of R462Q.

• Journal of Korean society of Dental Hygiene
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• v.17 no.1
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• pp.13-25
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• 2017

Objectives: The purpose of this study was to compare colony forming unit (CFU) method and multiplex real-time polymerase chain reaction (MRT-PCR) method for accurate quantitative analysis of bacteria. Methods: We compared the CFU method and the MRT-PCR method, which are still used in Korea, for Prevotella intermedius (P. intermedius), a periodontal disease pathogen selected by MRT-PCR, and Streptococcus mutans (S. mutans), a dental caries causative organism. The subjects of this study were 30 patients who visited the C dental hospital. Results: Total microorganisms in MRT-PCR method were significantly higher in both types of bacteria (p<0.05), since DNA of dead bacteria was also analyzed. This was because the periodontal dise(-) anaerobes, and even dead bacteria contain large amounts of toxic substances called LPS in the extracellular membrane, and fimbriae and pili, which are motility structures, still remain as a strong toxic substance in periodontal tissue. Conclusions: Therefore, in terms of the total amount of bacteria found, the MRT-PCR method will be a useful technique for searching all the bacteria in the oral cavity including live bacteria, as well as sterilization.

• International Journal of Oral Biology
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• v.41 no.3
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• pp.149-154
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• 2016

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase ${\beta}-subunit$ gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC $33478^T$. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.292-296
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• 2013

This study utilized an analysis method for detecting six microorganisms, such as Actinobacillus actinomycetemcomitans, Campylobacter rectus, Porphyromonas gingivalis, Tannerella forsythus, Treponema denticola, and Prevotella intermedia, triggering periodontal disease, using multiplex real-time polymerase chain reaction (PCR). The analysis including internal control was made by dividing the six species into two groups using four fluorescence dyes, and it was verified that there was no interference or cross-reaction between the target species and different kinds of oral microbial species. Qualitative and quantitative analyses were conducted on each microorganism in various samples, such as saliva and the plaque, using the multiplex real-time PCR and comparative analysis between periodontitis patients and healthy people, revealing obvious differences between them.

• Tuberculosis and Respiratory Diseases
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• v.85 no.1
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• pp.89-95
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• 2022

Background: With the introduction of Xpert MTB/RIF assay (Xpert), its incorporation into tuberculosis (TB) diagnostic algorithm has become an important issue. The aim of this study was to evaluate the performance of the Xpert assay in comparison with a commercial polymerase chain reaction (PCR) assay. Methods: Medical records of patients having results of both Xpert and AdvanSure TB/NTM real-time PCR (AdvanSure) assays using the same bronchial washing specimens were retrospectively reviewed. Results: Of the 1,297 patients included in this study, 205 (15.8%) were diagnosed with pulmonary TB. Using mycobacterial culture as the reference method, sensitivity of the Xpert assay using smear-positive specimens was 97.5%, which was comparable to that of the AdvanSure assay (96.3%, p=0.193). However, the sensitivity of the Xpert assay using smear-negative specimens was 70.6%, which was significantly higher than that of the AdvanSure assay (52.9%, p=0.018). Usng phenotypic drug susceptibility testing as the reference method, sensitivity and specificity for detecting rifampicin resistance were 100% and 99.1%, respectively. Moreover, a median turnaround time of the Xpert assay was 1 day, which was significantly shorter than 3 days of the AdvanSure assay (p<0.001). Conclusion: In comparison with the AdvanSure assay, the Xpert assay had a higher sensitivity using smear-negative specimens, a shorter turnaround time, and could reliably predict rifampin resistance. Therefore, the Xpert assay might be preferentially recommended over TB-PCR in Korean TB diagnostic algorithm.

• Tuberculosis and Respiratory Diseases
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• v.69 no.4
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• pp.250-255
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• 2010

Background: The purpose of this study was to evaluate recently developed real-time polymerase chain reaction (PCR) assay kit to detect Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) in respiratory specimens. Methods: We assessed the positive rate of the real-time PCR assay to detect MTB and NTM in 87 culture-positive specimens (37 sputum, 50 bronchial washing), which were performed real-time PCR by using $Real-Q_{TM}$ MTB&NTM Kit from January 2009 to June 2009, at Gyeongsang University Hospital. To compare the efficacy with the TB-PCR assay, we evaluated 63 culture-positive specimens (19 sputum, 44 bronchial washing) for MTB or NTM, which were performed TB-PCR by using ABSOLUTETM MTB II PCR Kit from March 2008 to August 2008. Results: Among 87 specimens tested using real-time PCR, MTB and NTM were cultured in 58 and 29, respectively. The positive rate of real-time PCR assay to detect MTB was 71% (22/31) and 92.6% (25/27) in AFB stain-negative and stain-positive specimens. For NTM, the positive rate of real-time PCR was 11.1% (2/18) and 72.7% (8/11) in AFB stain-negative and stain-positive specimens. Among 63 specimens performed using TB-PCR, MTB and NTM were cultured in 46 and 17, respectively. The positive rate of TB-PCR was 61.7% (21/34) and 100% (12/12) in AFB stain-negative and stain-positive specimens. TB-PCR was negative in all NTM-cultured 17 specimens. Conclusion: TB/NTM real-time PCR assay is useful to differentiate MTB and NTM in AFB stain-positive respiratory specimens and it is as effective in detecting MTB with TB-PCR.