• Journal of Life Science
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• v.24 no.4
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• pp.361-369
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• 2014

Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.

• Journal of Sericultural and Entomological Science
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• v.39 no.1
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• pp.30-35
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• 1997

A rapid and sensitive detection of BmNPV contamination in silkworm rearing room was carried by Plymerase chain reaction(PCR). Silkworm nuclear polyhedra were dissolved for the extraction of viral DNA within 30 minutes followed by the treatment of alkaline solution. The combination of primers of NP3 and NP2 was superior in PCR to the other 7 primers applied. Each primer was designed with 20 base in size and Newly designed NP3 of sense and the already reported NP2 for antisense were better in reaction than other primers. PCR products appeared 500bp in size. And annealing was confirmed proper at 55$^{\circ}C$ condition. Amplifiable template DNA amount was confirmed at least 100 ng to 0.1 ng and regarded as applicative for the assay of silkworm rearing environmental condition of sericultural farm. In case of the detection of BmNPV from the dust, sensitivity by PCR was as high as 1,000,000 times than that of microscopic observation.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• Journal of Microbiology
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• v.44 no.1
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• Korean Journal of Plant Taxonomy
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• v.46 no.2
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• pp.163-174
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• 2016

Gnetum (Gnetaceae, Gnetales) is a gymnosperm genus with ca. 35 species distributed in tropical forests around the world. Due to its dioecious habit and lack of diagnostic characters from vegetative tissue, the identification of Gnetum species is not easy without seeds or reproductive structures. To identify and verify their phylogenetic positions, we applied DNA barcoding to Cambodian Gnetum collections gathered between 2010 and 2015, with previously designed cp matK gene primers. We newly sequenced partial matK sequences from 72 Gnetum collections, 43 out of 72 from Cambodia, and analyzed 115 Gnetum accessions using the neighbor-joining method. The resulting neighbor-joining tree categorized Cambodian Gnetum samples into three clades of species: G. macrostachyum, G. montanum, and G. aff. gracilipes. The recognition of G. aff. gracilipes in Cambodia is reported here for the first time. Taxonomic information for the three recognized Cambodian Gnetum species is provided and the benefits of the taxonomic reevaluation assisted by DNA barcoding are emphasized in this work.

• Research in Plant Disease
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• v.14 no.3
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• pp.233-237
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• 2008

Tomato yellow leaf curl virus (TYLCV), a newly reported Geminivirus from tomato, generated recently large economic losses in Korea. Development of a fast and precise genetic diagnosis technique for detecting TYLCV which Agricultural research and extension services can utilize easy and handy is very important to prevent yield losses. Virion Capture (VC)/PCR is a simple, accurate and economical genetic detection method without any works or commercial kits for the extraction of the nucleic acid from the infected plants. Primers of twenty two for detection of TYLCV were designed and tested with extracted total DNA or crude sap from tomato leaf infected with TYLCV and healthy plant. Nine primers for total DNA using conventional PCR and another 9 primers for VC/PCR were selected eventually. Primers of six having same specificity were selected from the two methods and tested with other Geminivirus, Tobacco leaf curl virus (TLCV) by VC/PCR. Finally specific primers of four were selected for detection of TYLCV using VC/PCR, and Deng (540, 541), a degenerate primer for Geminivirus reported in 1996, was also developed for VC/PCR.

• Research in Plant Disease
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• v.24 no.3
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• pp.233-236
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• 2018

A multiplex RT-PCR (mRT-PCR) assay was developed for the detection of the recently reported viruses, Cherry virus A (CVA), Little cherry virus 1 (LChV-1), and Little cherry virus 2 (LChV-2), in cherry plants in Korea. Eight sets of primers were designed for each virus and their specificity was tested by using various combinations of mixed primer sets. From the designed primer sets, one combination was selected and further evaluated to estimate the optimum temperature and detection limits of the mRT-PCR. A newly developed mRT-PCR assay was also tested using 20 cherry samples collected in the field. This mRT-PCR assay may be a useful tool for field surveys of diseases and the rapid detection of these three viruses in cherry plants.

• Journal of Life Science
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• v.21 no.7
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• pp.947-952
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• 2011

A number of epidemiological studies have identified human papillomavirus (HPV) types 1, 2, 3, 4, 7, 10, 27, 57, and 65 in cutaneous common warts. However, identification of the HPV subtype by conventional polymerase chain reaction (PCR) is time consuming with its multi-step laboratory process. In this study, we aim to develop a specific one-step multiplex polymerase chain reaction method which capably identifies six different HPV genotypes related to common warts. By HPV DNA sequence analysis, 6 pairs of specific primers were designed from the intergenic regions of genes L1 to E6, and from genes E2 to L2. DNA sequence analysis with the L1 gene sequence of the sample was performed to measure the specificity of multiplex PCR. HPV-1, -2, -3, -4, -27, and -57 were identified without cross amplification in 109 out of 129 samples. The sensitivity and specificity of our set of primers in detecting HPV were 85% and 99.5%, respectively. For the 20 samples where HPV type was not identifiable by our batch of primer sets, multiplex PCR with an additional set of HPV primers was done, where 7 were found positive for HPV-7 or -65. Our results demonstrate that the newly designed multiplex PCR can rapidly detect the specific HPV subtype involved in common warts with high accuracy.

• Korean Journal of Veterinary Research
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• v.60 no.3
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• pp.109-116
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• 2020

This study aimed to develop a semi-nested polymerase chain reaction assay for the direct detection of Mycoplasma synoviae (M. synoviae) from clinical samples using three newly designed oligonucleotide primers specific to the variable lipoprotein haemagglutinin (vlhA) gene and differentiate M. synoviae field strains based on a nucleotide deletion or the insertion of the proline-rich repeat (PRR) region of the vlhA gene. The developed semi-nested polymerase chain reaction (PCR) assay revealed positive results in 12 out of 100 clinical samples collected from chickens showing lameness and joint swelling. Six positive samples were selected randomly for sequencing, and sequence analysis revealed 96.3-100% nucleotide identities compared to the reference sequences. Phylogenetic analysis showed that sequences of the strains in this study were closely related to WVU1853 (Spain), CK.MS.UDL.PK.2014.2 (Pakistan), and F10-2AS (USA) strains, but they were distinct from the M. synoviae-H vaccine strain sequence. M. synoviae obtained from these samples were identified as types A and C with a length of 38 and 32 amino acids, respectively. These results indicated that the specific and sensitive semi-nested PCR could be a useful diagnostic tool for the direct identification of clinical samples, and the sequence analysis of the partial vlhA gene can be useful for typing M. Synoviae.

• Korean Journal of Veterinary Service
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• v.38 no.2
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• pp.101-106
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• 2015

Murine mycoplasmosis, caused by Mycoplasma (M.) pulmonis, is a prominent disease in rodent animals. The aim of this study was to develop a sensitive and specific PCR assay to detect M. pulmonis in animals and to assess the suitability of this assay for the detection of mycoplasmal infection in rats experimentally infected with M. pulmonis. A new PCR assay using the M. pulmonis-specific primer pairs MPul-F and MPul-R was developed. The primers and probe for the assay were designed from regions in the 16S rRNA gene that are unique to M. pulmonis. The novel PCR assay was very specific and sensitive for M. pulmonis, detecting the equivalent of 5 pg of target template DNA. It detected only M. pulmonis and no other Mycoplasma species or other bacterial species. The newly developed PCR assay also effectively detected M. pulmonis infection in rats. These results suggest that this PCR assay using M. pulmonis-specific primer pairs of MPul-F and MPul-R will be useful and effective for monitoring M. pulmonis infection in animals.