• International Journal of Oral Biology
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• 제44권3호
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• pp.96-100
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• 2019

The purpose of this study was to develop Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the 16S ribosomal RNA (16S rDNA) gene. The specificity of the primers was determined by conventional PCR using 29 strains of 27 oral bacterial species including P. mikwangii. The sensitivity of the primers was determined by qPCR using the purified genomic DNA of P. mikwangii KCOM $1628^T$ (40 ng to 4 fg). The data showed that the qPCR primers (RTB134-F4/RTB134-R4) could detect P. mikwangii strains exclusively and as little as 40 fg of the genomic DNA of P. mikwangii KCOM $1628^T$. These results suggest that the developed qPCR primer pair can be useful for detecting P. mikwangii in epidemiological studies of oral bacterial infectious diseases.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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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.

• Food Science and Biotechnology
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• 제18권5호
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• pp.1118-1123
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• 2009

Event-specific real-time polymerase chain reaction (PCR) detection method for genetically modified (GM) maize MIR604 was developed based on integration junction sequences between the host plant genome and the integrated transgene. In this study, 2 primer pairs and probes were designed for specific amplification of 100 and 111 bp DNA fragments from the zSSIIb gene (the maize endogenous reference gene) and MIR604. The quantitative method was validated using 3 certified reference materials (CRMs) with levels of 0.1, 1, and 10% MIR604. The method was also assayed with 14 different plants and other GM maize. No amplification signal was observed in real-time PCR assays with any of the species tested other than MIR604 maize. As a result, the bias from the true value and the relative deviation for MIR604 was within the range from 0 to 9%. Precision, expressed as relative standard deviation (RSD), varied from 2.7 to 10% for MIR604. Limits of detections (LODs) of qualitative and quantitative methods were all 0.1%. These results indicated that the event-specific quantitative PCR detection system for MIR604 is accurate and useful.

• Korean Journal of Radiology
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• 제21권7호
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• pp.925-928
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• 2020

• Pediatric Infection and Vaccine
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• 제23권3호
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• pp.194-201
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• 2016

목적: 폐렴구균은 주요 비인두 상재균으로, 주위 조직을 침범하여 침습성 감염을 일으킬 수 있어 보균율에 대한 감시가 중요하다. 본 연구에서는 임상에서 비인두 흡인물로부터 추출하고 남은 RNA를 이용하여 폐렴구균을 확인할 수 있는 실시간 중합효소 연쇄반응(real-time reverse transcription polymerase chain reaction [RT-PCR])법을 구축하고, 보균율 측정에 있어서의 정확성과 이점을 확인하고자 하였다. 방법: 2014년 9월부터 10월까지 중앙대학교병원에 입원하여 호흡기 바이러스 RT-PCR 검사를 시행받은 18세 이하의 소아들로부터 비인두 흡인물을 채취하였다. 먼저 배양법과 genomic DNA (gDNA)를 이용한 real-time PCR을 시행하여 폐렴구균 검출률의 정확성을 확인하였다. 이 중 처음 20개의 검체를 이용하여, 고전적인 배양법과 gDNA를 이용한 real-time PCR, 그리고 RNA를 이용한 real-time RT-PCR법을 시행하고 이를 비교 분석하였다. 결과: 총 157개의 검체에서 시행한 real-time PCR 검사는 기존의 배양검사와 일치율이 0.922 (P<0.01, Fisher exact test)로 매우 높았다. 배양검사에서 음성인 133개의 검체는 real-time PCR에서도 모두 음성을 보였다. 24개의 배양 양성 검체 중 21개의 검체는 real-time PCR에서도 양성이었지만, 나머지 검체는 음성 결과를 보였다. 20개의 검체에서 시행한 real-time RT-PCR 검사는 1개 검체를 제외하고 배양법 및 real-time PCR과 결과가 일치하였다. 한편, 배양법을 시행하고 결과를 확인하기까지는 총 26.5시간, real-time RT-PCR 검사에는 총 4.5시간이 소요되었다. 결론: 본 연구는 비인두 집락균 확인을 위한 real-time RT-PCR법의 확립과, 폐렴구균 보균율 측정에 있어서의 real-time RT-PCR 검사의 정확성 및 편의성을 보여주었다. Real-time RT-PCR 검사법은 주요 세균들의 보균율 연구에 있어서 시간과 노력을 줄일 수 있는 좋은 방법이며, 폐렴구균의 역학자료 수집에 큰 도움이 될 것으로 기대한다.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2016년도 제50회 동계 정기학술대회 초록집
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• pp.375.1-375.1
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• 2016

Polymerase chain reaction (PCR) has revolutionized genetics and become one of the most popular techniques in modern biological and medical sciences. It can be used not only as an in vitro DNA amplification method but also used in many bioassay applications. The PCR can be used to exponentially produce a large number of DNA copies from a small quantity of DNA molecules in a few hours. However, as unwanted DNA fragments are also often manufactured, the amplification efficiency of PCR is decreased. To overcome this limitation, several nanomaterials have been employed to increase the specificity of the PCR reaction. Recently, graphene has attracted a great interest for its excellent electron transfer, thermal and biocompatibility. Especially, gold nanoparticle-coated graphene oxide (GO/AuNPs) led to enhance electron and thermal transfer rate and low-charge transfer resistance. Therefore, we report the development of a demonstration for the PCR efficiency using a large-scale production of the GO and combination of gold nanoparticles. Because a thermal conductivity is an important factor for improving the PCR efficiency in different DNA polymerases and different size samples. When PCR use GO/AuNPs, the result of transmission electron microscopy and real-time quantitative PCR (qPCR) showed an enhanced PCR efficiency. We have demonstrated that GO/AuNPs would be simply outperformed for enhancing the specificity and efficiency of DNA amplification procedure.

• Tuberculosis and Respiratory Diseases
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• 제85권1호
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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.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제27권2호
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• pp.95-103
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• 2024

Purpose: Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. Methods: This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. Results: The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. Conclusion: The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient.

• Journal of Genetic Medicine
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• 제12권2호
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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.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제15권1호
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• pp.38-43
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• 2012

Purpose: Epstein-Barr virus (EBV) hepatitis is a usually asymptomatic and self-limiting disease in immunocompetent patients. However, the range of severity is wide, and the serological diagnosis is typically difficult until the convalescent phase. Thus, we examined the value of plasma EBV DNA real-time quantitative polymerase chain reaction (RT-qPCR) in EBV hepatitis for the timely diagnosis and the relationship between EBV viral load and clinical severity. Methods: Sixty samples were confirmed as having EBV infection by RT-qPCR with the EBV BALF5 gene sequence. We examined the clinical characteristics of EBV hepatitis by reviewing medical records. Results: The median total duration of fever was 8 days (range: 0-13 days). The mean peak value of aspartate aminotransferase (AST) was $241{\pm}214$ U/L, and the mean peak value of alanine aminotransferase (ALT) was $298{\pm}312$ U/L. There was no correlation between the serum levels of liver enzyme and plasma EBV DNA titer ($p$=0.1) or between median total duration of fever and EBV DNA titer ($p$=0.056). The median age of the EBV VCA IgM-negative group was lower compared with the EBV VCA IgM-positive group in EBV hepatitis (2 years vs. 6 years, $p$=0.0009). Conclusion: The severity of EBV hepatitis does not correlate with circulating EBV DNA load according to our data. Furthermore, we suggest that plasma EBV PCR may be valuable in young infants in whom the results of serology test for EBV infection commonly are negative.