• 대한수의학회지
• /
• 제36권1호
• /
• pp.119-129
• /
• 1996

The present study was conducted to rapidly detect Listeria monocytogenes in raw milk. Specificity and sensitivity of polymerase chain reaction(PCR) technique, and direct PCR were examinded in raw milk, also were compared the calssical culture methods with PCR technique. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L nomocytogenes. In the PCR specificity tests, each of the 10 strains of L monocytogenes tested gave a single 70-bp band. But the other six Listera spp tested gave negative results. Results of the sensitivity tests showed that as few as 2 CFU of L monocytogenes in pure cultures could be detected with 16S rRNA-based primers, L-1 and L-2. In different PCR cycles, a PCR product was detected with $10^3$ cells of L monocytogenes from 25 cycles to 50 cycles and the concentration of PCR products was cycle-dependent. Raw milk samopes added L monocytogenes cells gave negative results. However, these samplers gave a single 70-bp band by pretreatment of pronase, and PCR products were detected with $10^1$ cells of L monocytogenes. To detemine the most sensitive culture protocol to use in conjunction with the PCR assay, raw milk samples were inoculated with L monocytogenes at concentrations ranging from 1 to $5.7{\times}10^4CFU/ml$. PCR assays from Listeria enrichment broth(LEB) containing raw milk samples added L monocytogene EGD could dtect 10 cells in pronase-pretreated samples without incubation, and 1 cell of L monocytogenes in both 12 hr and 24 hr incubation, respectively. Isolation raw of PCR assays was similar to that of classical culture methods, but required time for detection of L monocytogenes could remarkably be reduced compare to culture methods.

• 대한화학회지
• /
• 제47권2호
• /
• pp.127-132
• /
• 2003

소의 타일레리아병 진단을 위해 DNA 추출과정 없이 전혈에서 바로 중합효소연쇄반응(PCR)을 통하여 T. buffeli(buffeli/orientalis/sergenti)의 16S rRNA 유전자 단편을 증폭시킨 뒤, 증폭된 DNA를 전기영동법으로 분석하는 방법을 개발하였다. 특이유전자 단편의 증폭을 위해 formamide를 사용하여 혈액세포를 용해시켰으며, 단백질의 응고를 줄이기 위해 낮은 반응온도를 사용하는 FoLT(Formamide Low Temp.) PCR법을 이용하였다. 전혈 100-200 nL를 바로 PCR 증폭에 사용하였으며, PCR 산물(816-bp DNA)은 전기영동법으로 분석하였다. 본 결과는 T. buffeli에 감염된 소의 혈액으로부터 정제된 DNA를 사용하여 얻은 실험결과와 잘 일치하였다.

• Animal cells and systems
• /
• 제13권3호
• /
• pp.351-356
• /
• 2009

Ciliates are undoubtedly one of the most diverse protozoans that play a significant role in ecology. However, molecular examination, based on comparing the DNA sequences, has been done on a limited number of the species. Because most ciliates are uncultivable and their population sizes are often too small, it is usually difficult to obtain sufficient genomic DNA required for PCR based experiments. In the present study, we evaluated the effectiveness of four commercial DNA extraction procedures that extract high quality genomic DNA from a single ciliate cell. It was discovered that RED Extract-N-$Amp^{TM}$ PCR kit is the best method for removing PCR-inhibiting substances and minimizing DNA loss during purification. This method can also amplify more than 25 reactions of PCR. In addition, this technique was applied to single cells of 19 species belonged to 7 orders under 5 classes that isolated from mixed natural populations. Their small subunit ribosomal DNA (SSU rDNA) was successfully amplified. In summary, we developed a simple technique for the high-yield extraction of purified DNA from a single ciliate cell that may be more useful for rare ciliates, such as tiny and uncultivable marine microbes.

• Fisheries and Aquatic Sciences
• /
• 제9권2호
• /
• pp.70-74
• /
• 2006

DNA sequence-based typing is considered a robust tool for the discrimination of dinoflagellate species because of the availability of extensive rDNA sequences. Here, we present a rapid, cost-effective DNA-sequencing technique for various PCR products. This sequencing strategy relies on 'nested' or 'tailed' primer labeled with near-infrared dye, and uses a minimal volume of unpurified PCR product (ca. $5{\mu}L$) as the DNA template for sequencing reactions. Reliable and accurate base identification was obtained for several hundred PCR fragments of rRNA genes. This quick, inexpensive technique is widely applicable to sequence-based typing in clinical applications, as well as to large-scale DNA sequencing of the same genomic regions from related species for studies of molecular evolution.

• 대한임상검사과학회지
• /
• 제39권3호
• /
• pp.161-166
• /
• 2007

To develop a rapid and reliable single-tube-based PCR technique for detection simultaneously the quinolone resistance qnrA, qnrB and qnrS genes. After multiple alignment, primers were designed to detect known qnr variants. I was used for A total of 43 extented-spectrum ${\beta}$-lactamases (ESBLs) producing Escherichia coli and Klebsiella pneumoniae isolated from university hospital were tested for screening, as with qnr genes. In optimized conditions, all positive controls confirmed the specificity of the PCR primers. Out of 43 isolates, qnrA genes were detected 19 (44.2%), qnrB genes 5 (11.7%), qnrS genes 15 (34.9%) and 8 (18.6%) isolates were not detected. I report here a fast and reliable technique for rapid screening of qnr positive strains to be used for epidemiological surveys.

• Tuberculosis and Respiratory Diseases
• /
• 제39권1호
• /
• pp.1-6
• /
• 1992

The diagnosis of tuberculosis is usually established using staining and culturing techniques. Fluorescent stains have improved the sensitivity of direct microscopy. Improved culture media coupled with radiometric means of detecting early mycobacterial growth have shortened the time needed for cultural diagnosis. Rapid immunodiagnostic techniques based on the detection of mycobacterial antigen or of antibodies to theses antigens have not, however, come into widespread clinical use. The DNA or RNA hybridization tests with labeled specific probes which have been described so far are not sensitive enough to be used for clinical speicimens without prior culturing. The advent of the polymerase chain reaction (PCR) has opened new possibilities for diagnosis of microbial infections. This technique has already been applied to a number of microorganisms. In the field of mycobacteria the PCR has been used to identify and to detect DNAs extracted from various mycobacteria. However, despite the extraordinary enthusiasm surrounding this technique and the considerable investiment, PCR has not emerged from the developmental "trenches" in the passed several years. It may be a considerable lenth of time before clinical microbiology laboratories become PCR playgrounds because many details remain to be worked out.

• 대한임상검사과학회지
• /
• 제37권3호
• /
• pp.139-142
• /
• 2005

Most expressed HLA(human leukocyte antigen) loci exhibit a remarkable degree of allelic polymorphism, which is derived from sequenceing differences predominantly localized to discrete hypervariable regions of the amino-terminal domain of the molecule. In this study, the HLA-DRB1 genotypes were determined in twenty students using the PCR-SSP (polymerase chain reaction-sequence specific primer) technique. Two specific primer pairs in assigning the DRB1 gene were used. The results of PCR-SSP, the $HLA-DRB1^{\ast}0101$ primer detected nine and $HLA-DRB1^{\ast}1501$ primer detected three people. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-DRB1 genotypes. Moreover, these genotype frequency results of the HLA DRB1 gene could be useful for database study before being applied to individual identification and transplantation immunity.

• Asian Pacific Journal of Cancer Prevention
• /
• 제17권2호
• /
• pp.491-495
• /
• 2016

Background: Small B-cell non-Hodgkins lymphoma (NHL) is difficult to be distinguished from non-neoplastic reactive processes using conventional haematoxylin-eosin (HE) staining due to different interpretations among pathologists with diagnosis based on morphologic features. Ancillary examinations such as immunohistochemical (IHC) staining are essential. However, negative or doubtful results are still sometimes obtained due to unsatisfactory tissue processing or IHC technique. The polymerase chain reaction (PCR) as a molecular diagnostic technique is very sensitive and specific. Clonality detection of heavy chain immunoglobulin (IgH) gene rearrangement has been widely used to establish diagnosis of B-cell NHL. Aims: To elaborate interobserver variation in small B-cell NHL diagnosis based on morphologic features only and to confirm sensitivity and specificity of the PCR technique as an ancillary method. Materials and Methods: A toptal of 28 samples of small B cell NHL and suspicious lymphoma were interpreted by 3 pathologists in Sardjito General Hospital based on their morphology only. The reliability of assessment and the coefficient of interobserver agreement were calculated by Fleiss kappa statistics. Interpretation results were confirmed with IHC staining (CD20, CD3, Bcl2). PCR was performed to analyze the clonality of IgH gene rearrangement. Results: Interobserver agreement in morphologic evalution of small B cell NHL and chronic lymphadenitis revealed kappa coefficient 0.69 included in the substantial agreement category. The cases were divided into 3 groups based on morphology and IHC results; lymphoma, reactive process and undetermined group. PCR analysis showed 90% sensitivity and 60% specificity. Conclusions: The present study revealed a substantial agreement among pathologists in small B-cell NHL diagnosis. For difficult cases, PCR is useful as complementary method to morphologic and IHC examinations to establish definitive diagnosis.

• 농업과학연구
• /
• 제38권1호
• /
• pp.45-50
• /
• 2011

This study was conducted to investigate the farm situation about bovine leukemia virus(BLV) infection that greatly influence productivity in dairy cattle and compare the accuracy of diagnosis for BLV infection between PCR and ELISA techniques. Blood samples of 193 heads from 5 herds in Chungnam and Chungbuk area were used to analyze BLV gene and serum, and the results were obtained as follows. The amplified BLV gene in dairy cattle by PCR technique resulted in 226 bp, 596 bp and 434 bp, respectively, for gag, pol and env, which were well amplified. The infection rates of BLV virus diagnosed by PCR and ELISA techniques ranged from 80.55 to 100% and from 22.22 to 86.95%, respectively, and the infection rates among 5 herds were significantly different in both methods (P<0.05). Further, the average infection rates of 5 herds were 87.05 and 63.21%, respectively, for PCR and ELISA techniques. Kappa statistics for examining consistency of diagnosis by PCR and ELISA techniques showed 0.246, which represents low consistency. Consequently, PCR based BLV technique was considered as a corrective measure for diagnosis of BLV infection in Holstein dairy cattle.

• Parasites, Hosts and Diseases
• /
• 제53권1호
• /
• pp.77-83
• /
• 2015

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.