• The Plant Pathology Journal
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• 제36권2호
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• pp.170-178
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• 2020

The Lily mottle virus (LMoV) impedes the growth and quality of lily crops in Lanzhou, China. Recently Arabis mosaic virus (ArMV) has been detected in LMoV-infected plants in this region, causing plant stunting as well as severe foliar symptoms, and likely posing a threat to lily production. Consequently, there is a need to develop simple, sensitive, and reliable detection methods for these two viruses to prevent them from spreading. Reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assays have been developed to detect LMoV and ArMV using two primer pairs that match six conserved sequences of LMoV and ArMV coat proteins, respectively. RT-LAMP assay results were visually assessed in reaction tubes using green fluorescence and gel electrophoresis. Our assays successfully detected both LMoV and ArMV in lily plants without the occurrence of viral cross-reactivity from other lily viruses. Optimal conditions for LAMP reactions were 65℃ and 60℃ for 60 min for LMoV and ArMV, respectively. Detection sensitivity for both RT-LAMP assays was a hundredfold greater than that of our comparative RT-polymerase chain reaction assays. We have also found this relatively rapid, target specific and sensitive method can also be used for samples collected in the field and may be especially useful in regions with limited or no laboratory facilities.

• Journal of Crop Science and Biotechnology
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• 제11권2호
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• pp.91-100
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• 2008

Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism(SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer(FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci(QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 $F_2$ plants from the cross of PI 96354$\times$Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot(FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 hours without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean.

• Asian-Australasian Journal of Animal Sciences
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• 제22권8호
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• pp.1107-1112
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• 2009

The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 $\mu{M}$ Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the Xsorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.248-255
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• 2012

In order to develop a protocol to quantify cyanobacteria and Microcystis simultaneously, the primers and probe were designed from the conserved regions of 16S rRNA gene sequences of cyanobacteria and Microcystis, respectively. Probe match analysis of the Ribosomal Database Project showed that the primers matched with over 97% of cyanobacterial 16S rRNA genes, indicating these can be used to amplify cyanobacteria specifically. The TaqMan probe, which is located between two primers, matched with 98.2% of sequences in genus GpXI, in which most Microcystis strains are included. The numbers of cyanobacterial genes were estimated with the emission of SYBR Green from the amplicons with two primers, whereas those of Microcystis spp. were measured from the fluorescence of CAL Fluor Gold 540 emitted by exonuclease activity of Taq DNA polymerase in amplification. It is expected that this method enhances the accuracy and reduces the time to count cyanobacteria and potential toxigenic Microcystis spp. in aquatic environmental samples.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7621-7628
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• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

• 한국약용작물학회지
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• 제25권6호
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• pp.389-396
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• 2017

Background: Panax ginseng C. A. Meyer is wood-cultivated ginseng (WCG) in Korea which depends on an artificial forest growth method. To produce this type of ginseng, various P. ginseng cultivars can be used. To obtain a WCG similar to wild ginseng (WG), this method is usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. Recently, the WCG industry is suffering a problem in that Panax notoginseng (Burk.) F. H. Chen or Panax quinquefolium L. are being sold as WCG Korean market; These morphological similarities have created confusion among customers. Methods and Results: WCG samples were collected from five areas in Korea. After polymerase chain reaction (PCR) amplification using the primer pair labeled with fluorescence dye (FAM, NED, PET, or VIC), fragment analysis were performed. PCR products were separated by capillary electrophoresis with an ABI 3730 DNA analyzer. From the results, WCG cultivated in Korea showed very diverse genetic background. Conclusions: In this study, we tried to develop a method to discriminate between WCG, P. notoginseng or P. quinquefolium using simple sequence repeat (SSR) markers. Furthermore, we analyzed the genetic diversity of WCG collected from five cultivation areas in Korea.

• 대한화학회지
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• 제55권2호
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• pp.262-267
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• 2011

형광 검출기를 기반으로 한 변성 고성능 액체 크로마토그래피(DHPLC)분석법은 핵산검출에 유용하게 사용되고 있으며 또한 등온증폭 반응법은 병원성 미생물의 효과적인 검출방법으로 알려져 있다. 이 연구에서는 HBV의 조기 분석 방법으로써 사용되는 등온증폭반응법(LAMP)과 변성고성능 액체 크로마토그래피(DHPLC)의 검출한계와 특이성, 그리고 민감도를 평가하였다. 등온증폭 반응법의 검출 시간이 가장 빠른 장점을 보였으나, 변성 고성능 액체 크로마토그래피 분석법이 등온증폭반응법과 real-time PCR분석법과 비교한 결과, 10배 이상의 높은 민감도를 확인 할 수 있었다. 본 결과로서 B형 간염 바이러스의 진단을 위하여, 빠른 검출법으로써 등온증폭 반응법이 유용하게 쓰일 수 있을 것이며 변성 고성능 액체 크로마토그래피 분석법은 좀 더 낮은 병원균의 감염도 검출할 수 있어, 임상에서 유용하게 사용할 수 있을 것이다.

• 한국응용곤충학회지
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• 제56권4호
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• pp.377-385
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• 2017

멸구과(Delphacidae) 8종의 internal transcribed spacer 2 (ITS2) DNA 염기서열로 종간 차이 추정값을 비교하고 분자계통수를 추론하였다. ITS2 DNA 염기서열 길이는 종(species)마다 550 bp (흰등멸구)에서 699 bp (겨풀멸구)까지 차이를 보였다. 같은 Nilaparvata 속의 겨풀멸구와 벼멸구붙이 사이의 염기서열 차이 추정값($d{\pm}S.E.$)은 $0.001{\pm}0.001$로 가장 낮았으며, 사슴멸구와 일본멸구 사이는 $0.579{\pm}0.021$로 가장 높았다. 벼멸구와 다른 멸구류들과의 종간 염기서열 차이 추정값은 $0.056{\pm}0.008$ (겨풀멸구)에서부터 $0.548{\pm}0.021$ (일본멸구)로 구분되었다. 반면, Neighbor-joining 방법으로 추론된 분자계통수에서는 겨풀멸구와 벼멸구붙이를 제외하고 나머지 멸구류들은 독립된 다른 그룹으로 분지되었다. 벼멸구의 ITS2 염기서열을 참고하여 벼멸구 특이 고리매개등온증폭(loop-mediated isothermal amplification, LAMP) 프라이머 4 세트(BPH-38, BPH-38-1, BPH-207 및 BPH-92)를 제작하였다. 이들 각각을 벼멸구, 흰등멸구 및 애멸구의 게놈 DNA와 $65^{\circ}C$에서 60분간 반응시켰을 때, 벼멸구 시료에서만 증폭 산물들이 관찰되었다. BPH-92 LAMP 프라이머 세트로 $65^{\circ}C$에서 벼멸구 DNA의 양(0.1 ng, 1 ng, 10 ng, 100 ng)과 반응시간(20분, 30분, 40분, 60분)을 달리하여 형광반응을 관찰하였을 때, 20분과 30분 반응에서는 100 ng 까지에서도 발광여부 구별이 어려웠다. 그러나 40분 반응에서는 10 ng 이상에서, 60분 반응에서는 0.1 ng 이상에서 발광여부가 명확히 구별되었다.

• 한국응용곤충학회지
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• 제57권4호
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• pp.393-399
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• 2018

고리매개등온증폭법(LAMP)으로 흰등멸구를 특이적으로 구별해낼 수 있는 프라이머 세트(WBPH-65)가 핵내 ITS2영역의 전체염기서열(KC417469.1)을 바탕으로 설계 제작되었다. WBPH-65는 총 6개의 프라이머, F3 (18 bp), B3 (18 bp), FIP (43 bp), BIP (40 bp), LF (21 bp), LB (25 bp)로 구성되는데, 전체 합한 길이가 165 bp이다. WBPH-65를 흰등멸구, 벼멸구 및 애멸구의 게놈 DNA와 $65^{\circ}C$에서 60분간 고리매개등온증폭 반응시켰을 때, 흰등멸구 시료에서만 증폭 산물들이 관찰되었다. $65^{\circ}C$에서 WBPH-65와 흰등멸구 게놈 DNA의 양과 반응시간을 달리하여 형광반응을 관찰하였을 때 40분 반응에서는 10과 100 ng DNA에서, 60분 반응에서는 0.01, 0.1, 1, 10, 100 ng DNA에서 발광여부가 명확히 구별되었다. 그러나 20분과 30분 반응에서는 준비된 모든 DNA 양에서 발광여부 구별이 어려웠다. 한편, WBPH-65에서 LF와 BF 프라이머를 뺀 경우 60분 반응에서는 벼멸구, 애멸구 뿐만 아니라 흰등멸구의 게놈 DNA에서도 발광되지 않았다. 본 연구 결과로부터 WBPH-65가 60분 이내 반응에서 흰등멸구를 특이적으로 구별하기 위해서는 6개의 프라이머가 모두 필요하며 최소한 벼멸구와 애멸구를 구별해낼 수 있음을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권8호
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• pp.4533-4537
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• 2013

Objective: To use microarray chip technology for screening of stem cell radiation related miRNAs in laryngeal squamous cell carcinoma; study and explore the relationship of miRNAs with radiosensitivity of laryngeal squamous cells. Method: After conventional culture and amplification of the laryngeal squamous carcinoma cell line Hep-2, CD 133+ cells were screened out with combination of isolated culture of stem cell microspheres and FACS for preparation of laryngeal cancer stem cells. After radiation treatment, miRNAs of laryngeal squamous carcinoma stem cells before and after radiation were enriched and purified. After microarray hybridization with mammalian miRNA and scanning of fluorescence signal, the miRNAs of laryngeal squamous carcinoma stem cells before and after radiation was subject to differential screening and clustering analysis. Real-time quantitative RT-PCR was used to verify part of the differentially expressed miRNAs. Results: 70 miRNAs related to laryngeal cancer stem cell radiation with 2-fold difference in expression were screened out, in which 62 were down-regulated and 8 were up-regulated. Fluorescent quantitative RT-PCR results were consistent with miRNAs chip results. Conclusion: Some miRNAs may be involved in self-regulation with laryngeal squamous carcinoma stem cell radiation.