• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1044-1050
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• 2014

Since there is no consensus about the most reliable assays to detect invasive aspergillosis from samples obtained by minimally invasive or noninvasive methods, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis. Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, $200{\pm}20g$) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. The A. fumigatus DNA detection sequence was detected in the following number of samples from 12 immunosuppressed, infected rats examined on the scheduled days: day 1 (0/12), day 3 (0/12), day 5 (6/12), and day 7 (8/12) post-infection. The sensitivity and specificity of the qRT-PCR assay was 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct (cycle threshold) cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal Ct cut-off value was 1.40 (AUC, 0.919). The GM assay was more sensitive than the qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1709-1715
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• 2021

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.

• 한국미생물·생명공학회지
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• 제45권2호
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• pp.87-92
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• 2017

첫 번째 에볼라 출혈열 발발은 1976년 콩고 민주 공화국과 수단에서 발생했으며, 이후 2014년서 아프리카에서 27,741건, 11,284건의 사망자가 발생했다. 발열은 Filoviridae 계열에 속하며 ssRNA 게놈을 가진 에볼라 바이러스에 의해 발생했다. 바이러스의 알려진 아형은 Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus 및 Zaire ebolavirus이다. 역사적으로 에볼라의 주요 발생 지역은 동부 및 중부 아프리카 열대 지방에서 발생했다. 서아프리카에서의 발발로 인해 전세계 사회에서 수많은 사망과 공포가 확산되었다. 효과적인 치료와 백신이 없는 상황에서 전염병을 관리하고 통제하는 가장 중요한 방법은 정확한 진단을 통해서이다. WHO(세계 보건기구)는 체외진단(IVD) 검사에서 에볼라의 선택과 사용에 관한 긴급 지침을 발표했다. RealStar Ebolavirus Screen RT-PCR 키트 1.0 (Altona), Liferiver-Ebola Virus (EBOV) 실시간 RT-PCR 키트, Xpert 에볼라 검사 및 ReEBOV 항원 검사를 통해 수많은 회사 및 연구 기관에서 진단을 받고 4가지 WHO 조달 승인 진단을 확인했다. 또한, 신속한 검사 키트 Rapid Diagnosis Test (RDT)와 같은 새로운 진단법이 현재 연구 중이다.

• Pediatric Infection and Vaccine
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• 제25권2호
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• pp.91-100
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• 2018

목적: 본 연구는 30일 이상 90일 미만의 발열이 있는 영아에서 경험적 항생제 사용에 미치는 요소를 조사하였다. 방법: 2010년 1월부터 2016년 12월까지 부산대학교 어린이병원에 발열이 있는 이전에 건강했던 영아를 대상으로 임상양상, 검사소견, 항생제 사용에 대해 의무기록을 후향적으로 분석하였다. 호흡기 바이러스는 다중 역전사 중합 연쇄반응검사로 검출하여 1-3일 후 보고되었고, enterovirus는 중합 연쇄반응검사로 검출하여 수시간 만에 보고되었다. 결과: 366명의 대상자 중 129명은 경험적 항생제를 사용하였고 237명은 경험적 항생제를 사용하지 않았다. 입원 전 발열기간이 긴 경우와 호흡기 증상이 있을 때, 아파보일 때, CRP 상승 시 경험적 항생제 사용이 많았다. 경험적 항생제를 사용하지 않은 환자의 재입원율이 낮았다. Enterovirus polymerase chain reaction (PCR)이 검출된 대부분의 환자는 경험적 항생제를 사용하지 않았다. Respiratory virus multiplex reverse transcriptase (RT)-PCR 결과는 경험적 항생제 사용에 차이를 보이지 않았다. 결론: 본 연구에서 respiratory virus multiplex RT-PCR은 항생제 처방에 영향을 주지 않았고 enterovirus PCR은 항생제 처방에 영향을 주었다. Respiratory virus multiplex RT-PCR 결과가 신속하게 보고된다면 항생제 사용에 영향을 줄 것이다.

• The Plant Pathology Journal
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• 제31권3호
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• pp.219-225
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• 2015

The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

• Molecules and Cells
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• 제40권10호
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• pp.796-804
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• 2017

Endogenous retroviruses (ERVs) have been integrated into vertebrate genomes and have momentously affected host organisms. Horses (Equus caballus) have been domesticated and selected for elite racing ability over centuries. ERVs played an important role in the evolutionary diversification of the horse genome. In the present study, we identified six equine ERV families (EqERVs-E1, I1, M2, P1, S1, and Y4), their full-length viral open reading frames (ORFs), and elucidated their phylogenetic relationships. The divergence time of EqERV families assuming an evolutionary rate of 0.2%/Myr indicated that EqERV-S3 (75.4 million years ago; mya) on chromosome 10 is an old EqERV family and EqERV-P5 (1.2 Mya) on chromosome 12 is a young member. During the evolutionary diversification of horses, the EqERV-I family diverged 1.7 Mya to 38.7 Mya. Reverse transcription quantitative real-time PCR (RT-qPCR) amplification of EqERV pol genes showed greater expression in the cerebellum of the Jeju horse than the Thoroughbred horse. These results could contribute further dynamic studies for horse genome in relation to EqERV gene function.

• The Plant Pathology Journal
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• 제35권2호
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• pp.164-171
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• 2019

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with realtime detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of $1{\times}10^4$ to $1{\times}10^{12}$ ASSVd RNA $copies/{\mu}l$ with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from $1{\times}10^7$ to $1{\times}10^{12}$ copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

• 한국해양생명과학회지
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• 제1권2호
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• pp.79-87
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• 2016

카드뮴과 같은 중금속은 독성이 높아 수서 생물과 인간에게 해로운 영향을 미친다고 알려져 있다. 본 연구에서는 해양 섬모충 Euplotes crassus에서 카드뮴이 해독 기전에 관여하는 ABC transporters (ABCs)와 glutathione S-transferase (GST)의 유전자 발현에 미치는 영향을 조사하였다. 총 7개의 ABCs 유전자와 1개의 GST 유전자 일부를 클로닝하여 유전자 분석을 실시하였고, 카드뮴(0.1~1 mg/l) 노출에 따른 이들 유전자의 발현 양상을 quantitative real time RT-PCR (qRT-PCR)을 이용하여 분석하였다. 염기서열 분석과 계통 분석 결과 이들 ABCs 유전자가 ABC transporter의 특징을 가지며, ABC-B/C family에 속하는 것을 확인하였고, GST 유전자는 theta isoform과 유사한 것으로 나타났다. 카드뮴에 8시간 노출시킨 결과 ABC transporter 유전자의 경우 ABCB21 유전자를 제외하고는 대부분 농도 의존적으로 유전자 발현이 유의하게 증가하였다. GST 유전자는 0.5 mg/l에서 가장 높은 유전자 발현 양상을 보였으며, 1 mg/l에서는 발현량이 대조군 수준으로 감소되었다. 본 연구 결과는 E. crassus의 ABC transporter와 GST 유전자가 카드뮴에 의해 유도되는 독성에 대한 방어 기전에 참여하는 것을 의미한다.

• BMB Reports
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• 제43권7호
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• pp.480-484
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• 2010

The Bombyx mori Microarray Database (BmMDB; http://silkworm.swu.edu.cn/microarray) provides information for tissue-specific gene expression by using the whole-genome oligonucleotide microarray in the silkworm. We analyzed the tissue-specific expression patterns in the silk gland, fat body, and midgut five days of fifth instar larvae during the development of B. mori. To verify the tissue-specific expression, analysis was conducted using quantitative Real-time RT-PCR and the highly expressed endogenous Actin RNA as an intrinsic reference. Finally, we confirmed five genes, (sw15872, sw00692, sw20990, sw05300,and sw2250), out of 18 candidates expressed in two different tissues, which was consistent with the data published by Dr. Xiang's group, thereby supporting the BmMDB. Further studies for promoter regions of candidate genes can be applied in creating transgenic silkworms as biomedical insects for use in producing biomaterials, and to serve as well-characterized models for understanding the mechanism for the genetic regulation of tissue-specific development.

• Asian-Australasian Journal of Animal Sciences
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• 제26권3호
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• pp.423-432
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• 2013

Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don't know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ${\beta}$-actin (ACTB), ${\beta}$-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese.