• Korean Journal of Agricultural Science
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• v.38 no.1
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• pp.45-50
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• 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
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• v.54 no.1
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• pp.1-8
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• 2016

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, $RIDA^{(R)}$ Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.70-76
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• 2011

PCR-based Weissella species-specific detection method was developed to apply for the discrimination of Korean and Chinese kimchi by detecting a Weissella species only found in Korean or Chinese kimchi. PCR primers were designed from the species-specific sequence in the recN gene of each species. The primers allowed the species-specific detection and identification of nine species in the genera Weissella, and were successfully applied to the detection of W. cibaria, W. confusa, W. koreensis, and W. soli in kimchi with 20 ng template DNA. W. cibaria, W. confusa, and W. koreensis were detected from the Korean kimchi samples tested but W. soli was not detected. However, the four species were detected from Chinese kimchi samples. PCR-based W. soli-specific detection could not be perfectly applied as the Chinese kimchi discriminating method but has significance as an approach to evaluate the potential of scientific verification method based on the difference of microbial community.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.745-752
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• 2015

Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from non-tuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/discrimination of this bacterium from clinical samples.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.685-689
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• 2000

Due to the uncultivable nature of Mycobacterium leprae in vitro, the fast, easy, and accurate measurement of the antimicrobial drug susceptibility of this microbe has been difficult. Conventional methods for such testing are subjective, cumbersome, and expensive in some cases. Here, the utility of a reverse transcriptase-PCR (RT-PCR)-based assay for testing was examined and compared with a Buddmeyer-type radiorespirometric assay. The susceptibility of M. leprae to rifampin was determined by probing the presence of M.leprae-specific 18 kDa gene mRNA in M. leprae-infected IC-21 macrophage cells after drug treatment. The results showed that, as the refampin concentration was increased, the 360-bp cDNA products generated by the RT-PCR-based assay decreased in a dose-dependent manner as in the drug susceptibility observed in the Buddmeyer-type assay. The drug susceptibility testing of M. leprae by the RT-PCR based assay was found to be not only faster but also nearly $10^4$-fold more sensitive than the Buddmeyer-type assay. Moreover, it was also found that, unlike the RT-PCR based assay, the same testing by a DNA-PCR resulted in no differences in the 360-bp signal, regardless of the rifampin concentrations used. Accordingly, these results demonstrated that the drug susceptibility of M. leprae can be determined effectively by an RT-PCR-based assay, thereby providing a new, fast, and sensitive testing method.

• Journal of Veterinary Clinics
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• v.25 no.4
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• pp.241-244
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• 2008

This study examined the prevalence of Brucella spp. and Leptospira interrogans in 360 clinically healthy dogs using multiplex nested PCR. Four dogs (1.1%, 2 females and 2 males) tested positive to Brucella spp. by multiplex nested PCR. Fifty nine (16.4%, 31 females and 28 males) of 360 dogs tested positive L. interrogans. In 1 and 2 of the samples that tested positive to Brucella spp. and L. interrogans, the partial sequences of the virB1 and 16S rRNA genes were identified by direct sequence analysis, respectively. In conclusion, prevalence of Brucella spp. and L. interrogans by multiplex nested PCR revealed low and high, respectively. Multiplex nested PCR is can be useful for early detection of Brucella spp. and L. interrogans in the canine blood from asymptomatic dogs.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.67-75
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• 2003

A total of 260 dogs were randomly selected from two different treed kennels that brucellosis has occurred (group 1, 126 dogs), and random selected breed kennel (group 2, 134 dogs), and monitored for Brucella canis (B. canis) by 2-mercaptoethanol rapid slide agglutination test (2ME-RSAT) and bacterial culture method. For the differentiation, PCR-RFLP using omp-31, wbkA and per genes used for 52 of B canis strains (strain I) isolated in this study and 3 of B. canis strains (strain II) isolated in 1994 in Korea. 2ME-RSAT revealed that 63/126 dogs (50.0%) and 12/134 dogs (9.0%) were positive in group I and group II, respectively. Bacterial culture revealed that 47/126 dogs (37.3%) and 5/134 dogs (3.7%) were positive in group I and group II, respectively. As the results of PCR-RFLP, $\underline{omp}-31$ was amplified from all Brucella spp, except B. abortus. All B. canis isolates showed unique PCR-RFLP pattern following digestion with Bmel8I. However, all Brucella spp. showed the same PCR-RFLP pattern following digestion with SalI. PCR-RFLP analysis of wbkA revealed that all Brucella spp. showed the same pattern following digestion with HindIII. PCR-RFLP analysis of per revealed that B. abortus 544 and B. melitensis 63/9 showed the same pattern, but different from B. suis and B. canis following digestion with HindIII.

• Korean journal of applied entomology
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• v.37 no.1
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• pp.23-30
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• 1998

Restriction fragment length polymorphism (RFLP) of a DNA has been a useful tool for analyzing genetic variation. This research was performed to establish an RFLP analytic method on the mitochondrial DNA (mtDNA) of the beet armyworm, Spodoptera exigua (Hiibner). To do this, total size of the mtDNA was measured and polymerase chain reaction (PCR) primers were selected. Its mitochondrial genome size was ca. 16kb. From a serial PCR test of 29 primers refered to the compilation of Simon et al. (1994), 22 primers were selected to amplify its mtDNA fragments. These primers resulted in short (300-700 bp) or long (1000-2000 bp) DNA products which represented a total or partial sequence of each of CO-I, CO-11, Cyt-B, ND-1, 12s rRNA, 16s rRNA, and some tRNAs. PCR-RFLP was performed in some variable mtDNA regions with 8 kinds of 4bp recognizing restriction enzymes. Different populations from Andong, Kyungsan, and Sunchun did not show any restriction site polymorphisms but had some length variation in certain regions of mtDNA.

• Parasites, Hosts and Diseases
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• v.50 no.2
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• pp.103-111
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• 2012

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.77-83
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• 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.