• Parasites, Hosts and Diseases
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• 제56권3호
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• pp.295-300
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• 2018

Human sparganosis was diagnosed by morphological and genetic analyses in Korea. The complete mitochondrial genomes of Spirometra erinaceieuropaei and S. decipiens isolated in Korea have been recorded. Present study was performed to provide information to diagnose the etiologic agent of sparganosis by multiplex PCR using mitochondrial genome sequences of S. erinaceieuropaei and S. decipiens. In an effort to examine the differential diagnosis of spirometrid tapeworms, multiplex PCR assays were performed on plerocercoid larvae of S. erinaceieuropaei and S. decipiens. The PCR products obtained using species-specific primers were positively detected in all PCR assays on mixture of S. erinaceieuropaei and S. decipiens DNA. S. erinaceieuropaei-specific bands (239 bp and 401 bp) were obtained from all PCR assays using a mixture of S. erinaceieuropaei-specific primers (Se/Sd-1800F and Se-2018R; Se/Sd-7955F and Se-8356R) and S. erinaceieuropaei template DNA. S. decipiens-specific bands (540 bp and 644 bp) were also detected in all PCR assays containing mixtures of S. decipiens-specific primers (Se/Sd-1800F and Sd-2317R; Se/Sd-7955F and Sd-8567R) and S. decipiens template DNA. Sequence analyses on these species-specific bands revealed 100% sequence identity with homologous regions of the mtDNA sequences of S. erinaceieuropaei and S. decipiens. The multiplex PCR assay was useful for differential diagnosis of human sparganosis by detecting different sizes in species-specific bands.

• Genomics & Informatics
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• 제19권3호
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• pp.34.1-34.6
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• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• Journal of Genetic Medicine
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• 제5권1호
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• pp.41-46
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• 2008

목 적 : 취약 X 증후군(fragile X syndrome)은 FMR1 유전자의 5' 비해독부위에 있는 CGG 3염기 반복의 확장에 의해 발생되는 유전성 질환이다. 방 법 : 본 연구에서는 임상 소견과 핵형분석에서 취약 X 증후군으로 진단 받은 여아 환자와 그 부모를 대상으로 Abbott Molecular Fragile X PCR Kit를 이용하여 CGG 3염기 영역을 PCR로 증폭하여 normal, premutation, full mutation의 CGG 반복의 유형을 확인하였으며, premutation과 normal allele의 경우에는 정확한 CGG 반복수를 분석하였다. 결 과 : 환자는 30회와 >200회의 CGG 3염기가 반복된 FMR1 대립유전자를 갖고 있는 것으로 확인되어 취약 X 증후군으로 진단되었다. 또한 환자의 어머니에서 30과 98회의 반복 allele을 확인함으로써, 이 환자의 full mutation allele은 모계의 premutation allele로부터 유래한 것임을 알 수 있었다. 결 론 : Abbott Molecular Fragile X PCR Kit를 사용한 진단방법은, 취약 X 증후군환자의 경우에서 통상적으로 시행되고 있는 PCR, MS-PCR, Southern blotting을 병행하는 방법에 비해 신속하고 정확한 분자유전학적 진단이 가능한 유용한 방법이라 생각된다.

• Parasites, Hosts and Diseases
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• 제56권1호
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• pp.25-32
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• 2018

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris-EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was $40parasites/{\mu}l$ for P. falciparum and $35.2parasites/{\mu}l$ for P. vivax, whereas for Sn-PCR the limit of detection was $1.6parasites/{\mu}l$ for P. falciparum and $1.4parasites/{\mu}l$ for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.3003-3007
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• 2015

Objective: To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Materials and Methods: Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). Results: The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p<0.05). Patients with early EOC had markedly lower serum CA125 than those with advanced EOC (p<0.05), but there was no significant difference in free DNA methylation (p>0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (p<0.05). In the detection of patients with advanced EOC, the PPV of CA125 detection was obviously lower than that of multiplex nested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Conclusions: Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.

• Parasites, Hosts and Diseases
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• 제52권4호
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• pp.377-381
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• 2014

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.

• 식물병연구
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• 제15권2호
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• pp.57-62
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• 2009

우리나라 벼에 발생하는 주요 바이러스 중 애멸구에 의하여 전염하는 벼줄무의잎마름병(RSV)과 벼검은줄오갈병(RBSDV)에 대한 간편한 유전자 진단법인 VCHT-PCR 방법을 개발하였다. 벼 잎의 경우 즙액 추출 완충액은 0.5% sodium sulfite를 첨가한 0.01 M 인산완충액(pH 7.0)을 기본 완충액으로 이용하고 애멸구를 진단할 경우에는 기본 완충액에 2% PVP을 첨가하였을 때 VC/RT-PCR 진단이 잘 되었다. VC/RT-PCR을 이용한 진단에 적합한 RSV와 RBSDV 프라이머를 선발하였고 이것을 이용하여 동시진단으로 경기도 김포, 평택, 시흥지역에서 채집한 애멸구의 RSV와 RBSDV의 보독충을 쉽고 경제적으로 진단 할 수 있었다. ELISA에 의한 진단결과와 비교할 때 세 지역을 합하여 RSV에 대한 평균 보독충률은 9.2%로 동일하였으나 보독충 중 일부가 ELISA와 VC/RT-PCR 두 방법에 의한 진단결과가 다르게 나온 것은 RSV의 혈청학적, 유전적 계통 존재 가능성을 제시하고 있다. 포장에서 채집한 애멸구의 VC/RT-PCR 진단효율은 RSV와 RBSDV를 동시에 진단하므로 써 RSV만 진단이 가능한 ELISA 결과 보다 3.2% 높았다.

• Parasites, Hosts and Diseases
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• 제51권1호
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• pp.1-8
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• 2013

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.

• 대한수의학회지
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• 제44권1호
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• pp.99-103
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• 2004

The aim of the present study was to develop a sensitive and specific assay for the diagnosis of alcelaphine herpesvirus 1 (AlHV-1) which is a cause agent of malignant catarrhal fever in ruminants. A1HV-1 is a gamma herpesvirus, which is frequent latent, and it is often difficult to detect its antigens or specific nucleic acids because of its low genomic copies in the infected tissues. In this study, polymerase chain reaction (PCR)-dot blot hybridization (DBH) assay for detecting AlHV-1 DNA was developed and evaluated for its sensitivity and specificity as comparison with PCR and DBH alone. The developed PCR-DBH was more sensitive than PCR or DBH alone and also very specific. The results showed that the sensitivity of PCR-DBH were higher and stronger than those of PCR and DBH alone. This PCR-DBH assay can be applied efficiently to confirm the presence of AlHV-1 virus on clinical samples and to differentiate specifically between AlHV-1 infection and other viral infections.

• Parasites, Hosts and Diseases
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• 제56권3호
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• pp.229-236
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• 2018

Cutaneous leishmaniasis (CL) has been one of the most common parasitic diseases in Saudi Arabia. This study exhibits the clinical features, diagnosis, cytokine profile and treatment of CL patients in Al-Taif province. Ninety CL suspects at a tertiary care general hospital were enrolled in one-year study. Patients were interviewed, clinically-examined, and subjected to laboratory tests: skin scraping smear microscopy, OligoC-TesT commercial PCR (Coris BioConcept) and kinetoplast DNA (kDNA) PCR for Leishmania diagnosis. Interferon-gamma (RayBio; Human $IFN-{\gamma}$ ) and nitric oxide (NO) levels in patients' sera were evaluated before treatment with sodium stibogluconate (pentostam) with 20-day intramuscular drug regimen. Positive rates of microscopy, commercial PCR and kDNA PCR were 74.4%, 95.5% and 100%, respectively. Patients came to hospital mostly in winter (45.0%). CL was frequently exhibited in Saudi patients (78.8%), male gender (70.7%), age <20 years (50.0%), rural-dwellers (75.5%) and patients with travel history (86.6%). Lesion was mostly single ulcer (93.3%), occurred in the face (67.7%). Upon pentostam treatment, 85.1% of ulcers showed rapid healing signs. Levels of $IFN-{\gamma}$ and NO were significantly higher in the healing than the non-healing cases (P<0.001). The kDNA PCR proved more sensitive than microscopy and OligoC-TesT commercial PCR. Our results open perspectives for $IFN-{\gamma}$ use as a biomarker predicting treatment response.