• 제목/요약/키워드: touchdown PCR

검색결과 8건 처리시간 0.021초

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea

  • Shin, Ji-Hun;Lee, Sang-Eun;Kim, Tong Soo;Ma, Da-Won;Chai, Jong-Yil;Shin, Eun-Hee
    • Parasites, Hosts and Diseases
    • /
    • 제54권5호
    • /
    • pp.631-636
    • /
    • 2016
  • This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in > $1{\times}10^3$ oocysts for C. parvum, > $1{\times}10^4$ cysts for G. lamblia, and > 1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

Type-specific Amplification of 5S rRNA from Panax ginseng Cultivars Using Touchdown (TD) PCR and Direct Sequencing

  • Sun, Hun;Wang, Hong-Tao;Kwon, Woo-Saeng;Kim, Yeon-Ju;Yang, Deok-Chun
    • Journal of Ginseng Research
    • /
    • 제33권1호
    • /
    • pp.55-58
    • /
    • 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.

Molecular Structure of PCR Cloned PHA Synthase Genes of Pseudomonas putida KT2440 and Its Utilization for Medium-Chain Length Polyhydroxyalkanoate Production

  • Kim, Tae-Kwon;Shin, Hyun-Dong;Seo, Min-Cheol;Lee, Jin-Nam;Lee, Yong-Hyun
    • Journal of Microbiology and Biotechnology
    • /
    • 제13권2호
    • /
    • pp.182-190
    • /
    • 2003
  • A new phaC gene cluster encoding polyhydroxyalkanoate (PHA) synthase I PHA depolymerase, and PHA synthase II was cloned using the touchdown PCR method, from medium-chain length (mcl-) PHA-producing strain Pseudomonas putida KT2440. The molecular structure of the cloned phaCl gene was analyzed, and the phylogenic relationship was compared with other phaCl genes cloned from Pseudomonas species. The cloned phaCl gene was expressed in a recombinant E. coli to the similar level of PHA synthase in the parent strain P. putida KT2440, but no significant amount of mcl-PHA was accumulated. The isolated phaCl gene was re-introduced into the parent strain P. putida KT2440 to amplify the PHA synthase I activity, and the recombinant P. purida accumulated mcl-PHA more effectively, increasing from 26.6 to $43.5\%$. The monomer compositions of 3-hydroxylalkanoates in mcl-PHA were also modified significantly in the recombinant P. putida enforcing the cloned phaCl gene.

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

  • Hawash, Yoursry;Ghonaim, M.M.;Al-Shehri, S.S.
    • Parasites, Hosts and Diseases
    • /
    • 제54권1호
    • /
    • pp.1-8
    • /
    • 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.

분석조건별 담수어류의 환경 DNA 메타바코딩 효율 비교: 필터, 추출 키트, 프라이머 조합 및 PCR 방법 (Efficiency Comparison of Environmental DNA Metabarcoding of Freshwater Fishes according to Filters, Extraction Kits, Primer Sets and PCR Methods)

  • 김근식;김근용;윤주덕
    • 생태와환경
    • /
    • 제54권3호
    • /
    • pp.199-208
    • /
    • 2021
  • 메타바코딩을 이용한 환경 DNA 분석은 검출 감도가 높아 어류의 생물다양성 평가 및 멸종위기종의 검출에 유용한 기술이다. 이번 연구는 메타바코딩을 이용해 우리나라 담수어류를 대상으로 높은 검출 효율을 보일 수 있는 적합한 분석방법을 확인하기 위해 4가지 분석조건별, 즉 필터(cellulose nitrate filter, glass fiber filter), 추출 키트(DNeasy® Blood & Tissue Kit, DNeasy® PowerWater Kit), 프라이머 조합(12S rDNA, 16S rDNA) 그리고 PCR 방법(conventional PCR, touchdown PCR)로 나타나는 Operational Taxonomic Units(OTUs) 수와 종 조성을 비교하였다. Glass fiber filter와 DNeasy® Tissue & Blood Kit를 이용해 추출한 시료는 12S rDNA와 16S rDNA 프라이머 조합에서 담수어류 OTUs가 가장 많이 검출되었다. 모든 분석조건 중 프라이머 조합에서만 조기어강(Class Actinopterygii) 평균 OTUs 수에서 통계적으로 유의한 차이를 보였고(Non-parametric Wilcoxon Signed Ranks Test, p=0.005), 담수어류 평균 OTUs 수는 유의하지 않았다. 종 조성 비교 결과 역시 프라이머 조합에서 유의한 차이를 보였고(PERMANOVA, Pseudo-F=6.9489, p=0.006), 나머지 분석조건에서는 유의한 차이를 보이지 않았다. NMDS 분석 결과 종 조성은 유사도 65% 기준에서 프라이머 조합에 따라 묶였고, 16S rDNA 프라이머 세트는 주로 멸종위기종인 모래주사(Microphysogobio koreensis), 꼬치동자개(Pseudogobio brevicorpus)가 기여하였고, 12S rDNA 프라이머 세트는 주로 일반종인 피라미(Zacco platypus), 꺽지(Coreoperca herzi) 등이 기여한 것으로 나타났다. 본 연구는 국내 하천에서 채취한 시료에 대한 메타바코딩을 이용한 종 다양성 분석의 기초정보를 제공한다.

Improvement of PCR Amplification Bias for Community Structure Analysis of Soil Bacteria by Denaturing Gradient Gel Electrophoresis

  • Ahn, Jae-Hyung;Kim, Min-Cheol;Shin, Hye-Chul;Choi, Min-Kyeong;Yoon, Sang-Seek;Kim, Tae-Sung;Song, Hong-Gyu;Lee, Geon-Hyoung;Ka, Jong-Ok
    • Journal of Microbiology and Biotechnology
    • /
    • 제16권10호
    • /
    • pp.1561-1569
    • /
    • 2006
  • Denaturing gradient gel electrophoresis (DGGE) is one of the most frequently used methods for analysis of soil microbial community structure. Unbiased PCR amplification of target DNA templates is crucial for efficient detection of multiple microbial populations mixed in soil. In this study, DGGE profiles were compared using different pairs of primers targeting different hypervariable regions of thirteen representative soil bacteria and clones. The primer set (1070f-1392r) for the E. coli numbering 1,071-1,391 region could not resolve all the 16S rDNA fragments of the representative bacteria and clones, and moreover, yielded spurious bands in DGGE profiles. For the E. coli numbering 353-514 region, various forward primers were designed to investigate the efficiency of PCR amplification. A degenerate forward primer (F357IW) often yielded multiple bands for a certain single 16S rDNA fragment in DGGE analysis, whereas nondegenerate primers (338f, F338T2, F338I2) differentially amplified each of the fragments in the mixture according to the position and the number of primer-template mismatches. A forward primer (F352T) designed to have one internal mismatch commonly with all the thirteen 16S rDNA fragments efficiently produced and separated all the target DNA bands with similar intensities in the DGGE profiles. This primer set F352T-519r consistently yielded the best DGGE banding profiles when tested with various soil samples. Touchdown PCR intensified the uneven amplification, and lowering the annealing temperature had no significant effect on the DGGE profiles. These results showed that PCR amplification bias could be much improved by properly designing primers for use in fingerprinting soil bacterial communities with the DGGE technique.

발정 주기중 흰쥐 자궁에서의 Luteinizing Hormone (LH)과 수용체 유전자 발현 (Expression of Luteinizing Hormone (LH) and Its Receptor Gene in Uterus from Cycling Rats)

  • 김성례;이성호
    • Clinical and Experimental Reproductive Medicine
    • /
    • 제26권3호
    • /
    • pp.383-387
    • /
    • 1999
  • Objective: There is increasing evidence for the expression of rat in gene in several extrapituitary sites including testis and ovary. We also have demonstrated that the local LH expression in the rat epididymis and uterus, the major accessory sex organs in male and female reproductive system, respectively. Design: The present study was undertaken to elucidate whether the gene for LH receptor is expressed in rat uterus and whether the expressions of uterine LH and its receptor are differentially regulated during estrous cycle. Presence of the transcripts for rat LH receptor in the rat uterine tissue were confirmed by touchdown reverse transcription-polymerase chain reaction (RT-PCR). Results: In $LH{\beta}$ semi-quantitative RT-PCR, the highest expression level was shown in estrus stage. The level of ill receptor transcripts was also fluctuated during estrous cycle. In ovariectomized rats (OVX + Oil), the expressions of both uterine LH and LH-R were markedly reduced when compared to those from normal rats. Supplement with estradiol $17{\beta}$ to the ovariectomized rats (OVX + $E_2$) restored the expression levels of LH and its receptor to the levels in uteri from normal rats. Conclusion: Our findings indicated that 1) LH and its receptor gene are expressed in the rat uterus from cycling rats, 2) the expression of uterine LH and its receptor is mainly, if not all, under the control of ovarian sex steroid(s). These results suggested that the uterine LH may act as a local regulator with auto and/or paracrine manner, though the posibility that the pituitary LH may act directly on the regulation of uterine functions could not be discarded.

  • PDF

Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil

  • Yang, Wenjuan;Xu, Li;Zhang, Houjin;Yan, Yunjun
    • Journal of Microbiology and Biotechnology
    • /
    • 제25권11호
    • /
    • pp.1880-1893
    • /
    • 2015
  • In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/β hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60℃ with a Km of 0.37 mM and a kcat of 6.42 s-1. It retained over 90% of its original activity after incubation at 50℃ for 12 h. In addition, LipR was activated by Ca2+, Mg2+, Ba2+, and Sr2+, while strongly inhibited by Cu2+, Zn2+, Mn2+, and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.