• 한국축산식품학회지
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• 제35권3호
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• pp.382-388
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• 2015

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex realtime PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

• 한국균학회지
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• 제41권1호
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• pp.52-55
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• 2013

Phoma glomerata는 식물 잎이나 열매에 병을 일으키는 식물병원균으로 알려져 있다. 국내에서는 아직 피해사례가 없기 때문에 P. glomerata는 국내의 식물검역균으로 관리되고 있다. 본 연구는 국내에 들어오는 목재나 과일에 P. glomerata를 검출할 수 있는 방법 개발코자 수행되었다. Phoma 균주들의 translation elongation factor 1 alpha 유전자 염기서열에 기초하여 P. glomerata 특이적 PCR 프라이머를 디자인 하였고 그 특이성을 검정하였다. PCR 수행 결과 P. glomerata에서만 170 bp 크기의 밴드가 증폭되었고, 다른 비교 균주에서는 밴드가 증폭되지 않았다. 검출 감도를 평가하기 위해 기존 PCR방법과 real time PCR 방법을 이용하여 실험한 결과 최소 10 pg과 1 pg까지 각각 검출할 수 있었다. 본 연구결과는 디자인된 PCR 프라이머가 P. glomerata를 특이적으로 검출하는데 유용할 것임을 보여준다.

• Parasites, Hosts and Diseases
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• 제53권2호
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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.

• Genomics & Informatics
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• 제21권4호
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

• Asian-Australasian Journal of Animal Sciences
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• 제19권10호
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• pp.1503-1507
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• 2006

Goats' milk adulteration with cows' milk is becoming a big problem. In the past, the urea-polyacrylamide gel electrophoresis assay with different motility of ${\alpha}S1$-casein has been applied for the identification of cows' milk adulteration. The detection sensitivity is 1.0%. The aim of this study was to develop a faster and more sensitive method to detect cows' milk which may be present in adulterated goats' milk and goats' milk powder. The published primer was targeted at highly conserved regions in bovine mitochondrial DNA (a 271 bp amplicon). This amplicon was cloned and sequenced to further confirm bovine specific sequence. The chelex-100 was used to separate bovine somatic cells from goats' milk or goats' milk powder samples. Random sampling of different brands of goats' milk powder and tablets from various regions of Taiwan showed the adulterated rate was 20 out of 80 (25%) in goats' milk powders and 12 out of 24 (50%) in goats' milk tablets. With this system, as low as 0.1% cows' milk or cows' milk powder in goat milk or goat milk powder could be identified. This chelex DNA isolation approach provides a fast, highly reproducible and sensitive method for detecting the adulteration of goats' milk products.

• 식물병연구
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• 제23권1호
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• pp.65-68
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• 2017

2단계의 nested PCR 방법을 이용하여 보리 및 벼 재배 토양에서 Barley yellow mosaic virus (BaYMV)를 검출하였다. BaYMV 분절 RNA1 외피단백질 영역의 특이 프라이머로 1차 PCR을 하고 내부서열로부터 작성된 프라이머로 2차 PCR을 실시하여 확보된 372 bp의 PCR 산물이 BaYMV 외피단백질 영역과 98%-100% 염기서열이 일치하여 BaYMV를 검출할 수 있음을 확인하였다. 이 결과는 토양으로부터 BaYMV 검출에 관한 최초의 보고이며 토양전염성 바이러스의 정확한 진단과 예찰에 적용될 수 있을 것으로 생각한다.

• Journal of Ginseng Research
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• 제33권1호
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• pp.55-58
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• 2009

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1164-1169
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• 2008

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus-negative specimens. Furthermore, the assays could detect and subtype up to $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

• Parasites, Hosts and Diseases
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• 제49권4호
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• pp.381-384
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• 2011

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial largesubunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.

• Parasites, Hosts and Diseases
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• 제56권4호
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• pp.371-374
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• 2018

A 3-month-old female Maltese puppy was hospitalized with persistent diarrhea in a local veterinary clinic. Blood chemistry and hematology profile were analyzed and fecal smear was examined. Diarrheal stools were examined in a diagnostic laboratory, using multiplex real-time polymerase chain reaction (PCR) against 23 diarrheal pathogens. Sequence analysis was performed using nested PCR amplicon of 18S ribosomal RNA. Coccidian oocysts were identified in the fecal smear. Although multiplex real-time PCR was positive for Cyclospora cayetanensis, the final diagnosis was Cystoisospora ohioensis infection, confirmed by phylogenetic analysis of 18S rRNA. To our knowledge, this the first case report of C. ohioensis in Korea, using microscopic examination and phylogenetic analysis.