• 대한바이러스학회지
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• 제27권1호
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• Journal of Veterinary Science
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• 제24권3호
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• pp.40.1-40.6
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• 2023

Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples.

• Pediatric Infection and Vaccine
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• 제16권1호
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• pp.47-53
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• 2009

목 적: 국내 임신부의 생식기와 직장에서 분리된 B군 연구균의 혈청형 분포를 분자생물학적인 방법으로 확인하고자 하였다. 방 법: 산전 진찰을 위해 고양시 소재 4개의 산부인과병원을 방문한 임신 35주 이상 임신부의 생식기와 직장에서 분리 되어 $-70^{\circ}C$에 보관 중이던 42개의 B군 연구균에서 DNA를 추출하고 혈청형 특이 PCR 과 염기서열 분석을 이용하여 혈청형을 결정하였다. 결 과: 42개 균주 모두에서 혈청형 특이 PCR 과 염기서열 분석을 통해 혈청형을 결정할 수 있었고 빈도순으로 III형이 12균주(28.6%), V형이 11균주(26.2%), Ia형이 11균주(26.2%), VI형이 4균주(9.5%), Ib형이 2균주(4.8%), II형이 2균주(4.8%)였다. 결 론: 국내의 후기 임신부에서 흔하게 분리되는 B군 연구균의 혈청형은 III형, V형, Ia형이었고 본 연구에서 이용한 혈청형 특이 PCR과 염기서열 분석은 앞으로도 관련 연구에 유용하게 사용할 수 있을 것으로 보인다.

• 한국미생물·생명공학회지
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• 제27권5호
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• pp.359-363
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• 1999

To prevent appearance of resistant mosquitoes against $\delta$-endotoxin of bacillus thuringiensis subsp. israelensis (Bti) in field, a mosquitocidal Bacillus thuringiensis strain 21-2(Bt21-2) producing a new type of $\delta$-endotoxin was isolated. The strain Bt 21-2 belongs to H serotype 43, B. thuringiensis subsp. guiyangiensis (Btg). The $\delta$-endotoxins from the strain Bt 21-2 and the strain Bti were a cuboid shape morphologically, but the $\delta$-endotoxin of the strain Bt 21-2 was composed of 150, 90 and 70kDa proteins on SDS-PAGE, and the antigenicity of $\delta$-endotoxin of the strain Bt 21-2 was different from that of the strain Bti on immunoblot. The $\delta$-endotoxin gene of the strain Bt 21-2 was not amplified with specific primers of $\delta$-endotoxin gene (cry4A and cry4B) of the strain Bti on PCR.

• 한국식품영양학회지
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• 제13권1호
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• pp.1-5
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• 2000

Detection for heat-labile enterotoxin(LT) of enterotoxigenic Escherichia coli(ETEC, EC81, O148:H28) and enterotoxin of enterotoxigentic Clostridium perfringents type A(CP, NCTC8238, Hobbs serotype 2) by use of a polymerase chain reaction (PCR) assay were positive reaction, which using LT gene-specific primers of ETEC with a detection limit equivalent from 100ng/${\mu}\ell$ to 1 pg of a DNA fragment of 417-bp in EC81 and enterotoxin gene-specific primers of CP with a detection limit equivalent from 100ng/${\mu}\ell$ to 10pg of a DNA fragment of 364-bp in NCTC8238. Detection for a LT gene of ETEC highly appeared 10-fold sensitivity than an enterotoxin gene of CP.

• 한국식품과학회지
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• 제32권6호
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• pp.1336-1340
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• 2000

서울등 5개지역에서 수거한 냉동식품 624건을 대상으로 Y. enterocolitica를 분리하였다. 분리대상 식품군 중 해산물가공품중에서 Y. enterocolitica의 분리율(9.1%)이 가장 높았고, 다음으로 식육가공품(7.7%), 만두류(3.5%), 기타 냉동식품(3.4%)의 순이었으며 냉동피자류(1.7%)는 분리율이 가장 낮았다. 그리고 상반기와 하반기의 분리율이 차이를 보였는데 각각 1.6%와 9.6%로 하반기가 상반기에 비하여 분리율이 6배가 높았다. 냉동식품에서 분리된 Y. enterocolitica 균주는 모두 35개(5.6%)였고, 분리균주의 serotype은 O:5(9균주)와 O:1,2(1균주)이었다. 이외의 나머지 25개 균주는 본 연구에서 사용한 항혈청에 응집반응을 보이지 않아 혈청형을 typing할 수 없었다. 그리고 이들 균주를 biotype한 결과 모두 비병원성인 biotype 1A이었으며 중합효소연쇄반응(PCR)으로 확인 결과 또한 모두 비병원성으로 판명되었다.

• Clinical and Experimental Pediatrics
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• 제55권11호
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• pp.424-429
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• 2012

Purpose: Methods for quick and reliable detection of Streptococcus pneumoniae are needed for the diagnosis of pneumococcal disease and vaccine studies. This study aimed to show that sequential multiplex polymerase chain reaction (PCR) is more efficient than conventional culture in achieving S. pneumoniae -positive results. Methods: Nasopharyngeal (NP) secretions were obtained from 842 pediatric patients admitted with lower respiratory infections at Severance Children's Hospital in Korea between March 2009 and June 2010. For identification and serotype determination of pneumococci from the NP secretions, the secretions were evaluated via multiplex PCR technique with 35 serotype-specific primers arranged in 8 multiplex PCR sets and conventional bacteriological culture technique. Results: Among the results for 793 samples that underwent both bacterial culture and PCR analysis for pneumococcal detection, 153 (19.3%) results obtained by PCR and 81 (10.2%) results obtained by conventional culture technique were positive for S. pneumoniae. The predominant serotypes observed, in order of decreasing frequency, were 19A (23%), 6A/B (16%), 19F (11%), 15B/C (5%), 15A (5%), and 11A (4%); further, 26% of the isolates were non-typeable. Conclusion: As opposed to conventional bacteriological tests, PCR analysis can accurately and rapidly identify pneumococcal serotypes.

• 대한바이러스학회지
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• 제28권2호
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• pp.147-155
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• 1998

Hantavirus is a genus of the Bunyaviridae family consisting following serotype groups: Hantaan, Seoul, Puumala, Prospect Hill, Thailand, Belgrade, Thotta palayam, Sin Nombre. Most of Hantavirus group have been associated with many clinically similar disease known collectively as hemorrhagic fever with renal syndrome (HFRS). Hantaan virus is the prototype of the genus hantavirus, originally isolated from Apodemus agrarius. Bat was found as a natural host for Hantaan virus in Lee's lab for the first time. Then, Hantaan-like virus was isolated Hantaan-like virus from bat. To identify hantaviruses that are present in Korea among bats, bats were collected from Jeong-Sun, Won-Joo, Chung-Ju and Hwa-Cheon area, RNA was isolated from lung and serum. RT-PCR was performed with a universal primer from M segment. Nested RT-PCR was carried out to differentiate Hantaan, Seoul and Puumala virus using serotype specific primers. As we expected, Hantaan viruses were detected in bats and Seoul virus was not detected. Interestingly, Puumala viruses were also detected in bats from Won-Ju, but not in other areas. Puumala virus is originally isolated from Clethrinomys glareolus, and cause light HFRS. Recently, Paradoxomis webbiana, a wild bird turn out to be a reservoir for Puumala virus in Korea. These data indicate that bat is a new natural reservoir of Puumala virus.

• 미생물학회지
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• 제48권3호
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• pp.175-179
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• 2012

아데노바이러스는 다양한 급성호흡기감염증을 유발하며, 대부분 영유아나 어린이, 면역기능이 저하된 환자에게서 주로 나타나는 것으로 알려져 있다. 본 연구에서는 2009년부터 2011년까지 경기지역 소아청소년과 및 내과에 내원한 급성호흡기 감염증 의심환자를 대상으로 호흡기아데노바이러스의 유행양상 및 혈청형 분포양상을 분석하였다. 총 1,622명의 급성상기도 감염증이 의심되는 환자의 검체를 분석한 결과 102건(6.3%)에서 아데노바이러스를 검출하였다. 102건의 아데노바이러스 양성 검체에서 세포배양법으로 76주의 아데노바이러스를 분리하였고, 혈청형별 특이유전자인 헥손 유전자의 염기서열 분석을 통하여 혈청형을 확인하였다. 최근 3년간 경기도내에서 아데노바이러스 1형부터 6형까지 6개의 다른 혈청형이 분리되었고, 이 중 3형(n=40, 52.6%)이 가장 주류를 이루었다. 2009년에는 1형과 3형, 2010년에는 3형, 2011년에는 5형이 각각 우점하였다. 1, 2, 4, 5, 6형은 연중 산발적으로 확인되었으나, 3형은 산발적으로 발생하면서 2010년에는 큰 유행을 일으킨 것을 확인할 수 있었다. 본 연구결과를 통해 최근 3년동안 경기도내 아데노바이러스에 의한 outbreak의 주 원인 혈청형은 아데노바이러스 3형임을 알 수 있었고, 앞으로도 outbreak의 원인이 되는 특정 혈청형에 대한 지속적인 감시가 이루어져야 할 것이다.

• 대한수의학회지
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• 제44권3호
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• pp.399-405
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• 2004

Haemophilus somnus, Mycoplasma bovis and Pasteurella multocida were responsible for respiratory diseases in bovine. Methods for identifying these bacteria had poor sensitivity and specificity. In this paper, PCR assays were applied for rapid identification of H. somnus, M. bovis, P. multocida B:2 and P. multocida capsular types. The specific PCR products were amplified from H. somnus, but not from other bacteria. Ten-fold diluted H. somnus were mixed with P. multocida and then the mixed cultures were inoculated on agar plates. After incubation, PCR was performed with harvest from agar plates and could detect as few as 3.4 CFU/ml of H. somnus. The primers MboF and MboR produced an amplification product unique to M. bovis and sensitivity of PCR was as low as 100 pg of DNA. Only serotype B:2 of P. multocida, the causal agent of haemorrhagic septicemia in bovine, was specifically amplified in PCR among the 16 reference serotypes. The multiplex capsular PCR typing for P. multocida was produced the P. multocida specific product as well as the capsular serogroup-specific product. The present PCR assays should be useful for the rapid identification of bacterial pathogens from bovine respiratory diseases.