• Journal of Veterinary Clinics
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• v.23 no.3
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• pp.300-307
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• 2006

In the present study, methods of the reverse transcription-polymerase chain reaction(RT-PCR) were evaluated for the rapid detection and differentiation of transmissible gastroenteritis virus(TGEV), porcine epidemic diarrhea virus(PEDV) and rotavirus in piglets suffering from diarrhea. For the purposes, the PCR conditions were first confirmed for the amplification of VP7 gene of rotavirus and N gene of TGEV and PEDV using each specific primers and their annealing temperature. Multiplex RT-PCR methods were further determined to distinguish these viral infections and the results are as follows. For the specific amplification of these viral genes, the reliable PCR condition was determined as 30 cycles of reaction consisting each 1 min of denature at $94^{\circ}C$, annealing at $42^{\circ}C$ and polymerization at $72^{\circ}C$ with 1.0 mM $MgCl_2$. It was able to differentiate these viral infections in the intestines and feces of piglets suffering from diarrhea by duplex PCR for TGEV and PEDV and single PCR for rotavirus with a primer-annealing temperature of $42^{\circ}C$. When the multiplex RT-PCR were undertaken for the field samples, 17 cases of PEDV and 5 cases of rotavirus infections were differential diagnosed in a total of 92 samples of intestines and feces of the piglets with diarrhea.

• Korean Journal of Veterinary Research
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• v.42 no.2
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• pp.225-229
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• 2002

We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.

• The Korean Journal of Mycology
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• v.20 no.4
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• pp.315-323
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• 1992

The effects of several parameters on PCR amplification in using RAPD were studied. The results of this study suggest that approximately 15 ng of genomic DNA in $20\;{\mu}l$ of reaction mixture results in discrete and reproducible PCR products. In addition, the results indicate that concentration or amounts of reaction components studied are highly inter-dependent in their effects, and RNA can interfere severely with PCR amplification. Suitable concentrations or amounts of reaction components were found to be 30 ng of 10-mer primer, $200\;{\mu}M$ of dNTP, 0.001% gelatin 1.5 mM $MgCl_2$, 10 mM Tris-Cl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2 units of Taq DNA polymerase, and 15 ng of RNase-treated genomic DNA in $25\;{\mu}l$ of reaction mixture.

• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.499-505
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• 2018

Huanglongbing (HLB, Citrus greening disease) is one of the most devastating diseases that threaten citrus production worldwide. Although HLB presents systemically, low titer and uneven distribution of these bacteria within infected plants can make reliable detection difficult. It was known loop-mediated isothermal amplification (LAMP) method has the advantages of being highly specific, rapid, efficient, and laborsaving for detection of plant pathogens. We developed a new LAMP method targeting gene contained tandem repeat for more rapid and sensitive detection of Candidatus Liberibacter asiaticus (CLas), putative causal agent of the citrus huanglongbing. This new LAMP method was 10 folds more sensitive than conventional PCR in detecting the HLB pathogen and similar to that of real-time PCR in visual detection assay by adding SYBR Green I to mixture and 1% agarose gel electrophoresis. Positive reactions were achieved in reaction temperature 57, 60 and $62^{\circ}C$ but not $65^{\circ}C$. Although this LAMP method was not more sensitive than real-time PCR, it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Thus, we expect that this LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting the CLas in citrus and can be applied for rapid diagnosis is needed.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.19-25
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• 2006

With the use of Agrobacterium and gene-gun, cold regulated gene (BN115) has been injected in Chrysanthemum leaf disc and transgenic plants have been produced successfully on the selection media containing phytohormone. To determine the presence of the transferred cold regulated gene (BN115) in the transgenic Chrysanthemum, PCR-amplification indicated the presence of that gene. Real-Time PCR for confirmation of the putative transgenic plants was established. The copy number of cold regulated gene (BN115) is extrapolated on the basis of a standard curve. Serial dilutions of known number of gene copies were in triplicates. In this diagram, PCR cycles are plotted against the fluorescence intensity. The cycle at which the fluorescence reaches a threshold cycle is inversely proportional to the starting amount of target DNA.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.199-205
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• 2013

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

• Research in Plant Disease
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• v.23 no.1
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• pp.65-68
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• 2017

Barley yellow mosaic virus (BaYMV), which is transmitted by the root-inhabiting protist Polymyxa graminis, causes a soil-borne disease. In this study, we detected BaYMV from soil using two-step nested polymerase chain reaction (PCR). Specific primers based on a coat protein region of BaYMV segment RNA1 were used in the first round of amplification. Based on the sequenced amplicon, an inner primer was designed for the second round of amplification. A PCR product of 372 bp exhibited 98%-100% nucleotide sequence identity with the coat protein region of BaYMV segment RNA1. In this study, we propose an easy method for the detection of BaYMV from soil, may considerably assist in accurate fungus-transmitted virus diagnosis and subsequent disease forecasting. This is the first report on the detection of BaYMV from soil.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.56-62
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• 2000

Pasteurella multocida is kind of commensal bacteria in the upper respiratory tract of pigs. It is classified toxigenic and nontoxigenic strains based on the production of dermonecrotic toxin. Toxigenic strain is most associated with atrophic rhinitis which brings great economical loss in swine industry. However, toxigenic and nontoxigenic strains do not differ by diagnostic biochemical reaction or morphology. One of recently developed techniques, PCR detects the toxigenic P multocida. Amplification of an 846-nucleotide fragment of toxA gene was developed. The fragment amplified by PCR was detected in P multocida type D not type A. The PCR amplification was as sensitive as it could detect 1 pg of P multocida DNA. We compared the result of the PCR with the enzyme linked immunosorbent assay (ELISA) in a test for 40 swine nasal swabs. All of these isolates were toxin negative based on the ELISA while 2 isolates were detected in the PCR technique. in addition to accuracy, as required for rapid detection from contaminated nasal swabs, toxigenic P multocida was recovered efficiently from contaminated culture without inhibition of the PCR. The results show that the PCR detection of toxigenic P multocida directly form nasal swabs are feasible.

• Analytical Science and Technology
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• v.32 no.4
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• pp.155-162
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• 2019

Analysis techniques using DNA profiling are widely used in various fields including forensic science and new technologies such as the Direct PCR amplification method are being developed continuously in order to acquire the DNA profiles efficiently. However, it has a limits such as non-specific amplification according to the quality of crime scene evidence samples. Especially, split peaks caused by excessive DNA samples are one of the important factors that could cause the debate to allow researchers to interpret the DNA profile results. In this study, we confirmed the occurrence rate of split peaks in each STR (short tandem repeats) locus of the $GlobalFiler^{TM}$ kit and investigated the possibility of improving the split peaks using several PCR additives such as DMSO (dimethylsulfoxide), $MgCl_2$, Betaine and Tween-20. As a result, we could make three groups according to the occurrence rate of split peaks in Direct PCR and it was confirmed that the ratio of split peaks could be reduced by DMSO (87.4 %), $MgCl_2$ (84.5 %) and Betaine (86.1 %), respectively. These results indicate that PCR additives such as DMSO, $MgCl_2$ and Betaine can be improve the split peaks in Direct PCR and thereby facilitate subsequently a successful DNA profile results.