• 제목/요약/키워드: reverse blot hybridization assay

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자궁경부암 파라핀 조직에서 인유두종바이러스 유전형 검사의 유용성 평가 (Evaluation of Human Papillomavirus Genotyping from Formalin-fixed Paraffin-embedded Specimens in Cervical Cancers)

  • 진현우
    • 생명과학회지
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    • 제24권9호
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    • pp.1025-1029
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    • 2014
  • 자궁경부암은 전세계 여성의 사망원인 2위를 차지하며, 인유두종바이러스 감염과 상관성이 있다. 인유두종바이러스의 백신정책, 병인론, 추적관찰, 역학에서 유전형 검사는 중요하다. 후향적 연구를 위하여 파라핀 조직에서 역교잡반응을 이용하여 인유두종바이러스의 유전형을 검사하는 것은 명확하게 증명된 것은 아니다. 본 연구에서는 자궁경부암 파라핀 조직에서 역교잡반응을 사용하여 인유두종바이러스의 유전형 검사의 유용성을 평가하였다. 총 52개의 자궁경부암 파라핀 조직을 사용하여 역교잡반응을 실시하여 인유두종바이러스의 유전형을 검출하였다. 52개의 파라핀 조직중에서 32(61.5%) 건에서 인유두종바이러스가 검출되었으며, 단순감염이 27(84.4%) 건, 복합감염이 5(15.6%) 건으로 검출되었다. 단순감염에서 인유두종바이러스 유전형은 고위험군 18(8), 58(6), 16(5), 33(1), 35(1), 39(1), 56(1) 유전형과, 저위험군 11(2), 6(1), 70(1) 유전형으로 분석되었다. 복합감염에서 인유두종바이러스 유전형은 16/18(2), 18/52(1), 16/56(1), 16/18/33(1) 유전형으로 검출되었다. 본 연구를 통하여, 파라핀 조직으로 후향적 연구를 위한 인유두종바이러스 유전형 검사가 가능할 것으로 기대된다.

Mycobacterium tuberculosis DNA Detection and Molecular Drug Susceptibility Test in AFB-stained Sputum Slides

  • Jung, Dongju;Lee, Hyeyoung;Park, Sangjung
    • 대한의생명과학회지
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    • 제22권1호
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    • pp.24-28
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    • 2016
  • Tuberculosis (TB) remains an unsolved community health problem since identification of its causing microorganism called Mycobacterium tuberculosis (MTB) by Robert Koch in 1882. Annually, eight million TB cases are newly reported and 2~3 million patients die from TB. Pulmonary TB is highly infectious and untreated pulmonary TB patients are believed to infect >10 people in a year. The conventional methods for diagnosis of TB are chest X-ray and isolation of the causing microorganisms from patient specimens. Screening of TB is conducted with smeared sputum in slides, and TB is confirmed by identification of MTB in cultured specimens. One of the fatal pitfalls of screening detection for smeared sputum is that it is impossible to distinguish MTB and other acid-fast bacilli (AFB) because they are stained equally with Ziehl-Neelsen (ZN) stain. Culture of MTB is the most reliable method for diagnosis of TB but it takes 4~8 weeks. In this report, we suggest a fast and highly-reliable MTB detection method that distinguishes AFB in sputum samples. Purified DNA from the AFB stained slide samples offered by The Korean Institute of Tuberculosis were used to detect infected MTB in patients. PCR, real-time PCR and reverse blot hybridization assay (REBA) methods were applied to purified DNA. Conclusively, the real-time PCR method was confirmed to produce high sensitivity and we were able to further detect drug-resistant MTB with REBA.

Differentially Expressed Genes by Methylmercury in Neuroblastoma cell line using suppression subtractive hybridization (SSH) and cDNA Microarray

  • Kim, Youn-Jung;Chang, Suk-Tai;Yun, Hye-Jung;Ryu, Jae-Chun
    • 한국환경독성학회:학술대회논문집
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    • 한국환경독성학회 2003년도 춘계학술대회
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    • pp.187-187
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    • 2003
  • Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. In this study, two methods, cDNA Microarray and SSH, were performed to assess the expression profile against MeHg and to identify differentially expressed genes by MeHg in neuroblastoma cell line. TwinChip Human-8K (Digital Genomics) was used with total RNA from SH-SY5Y (human neuroblastoma cell line) treated with solvent (DMSO) and 6.25 uM (IC50) MeHg. And we performed forward and reverse SSH method on mRNA derived from SH-SY5Y treated with DMSO and MeHg (6.25 uM). Differentially expressed cDNA clones were sequenced and were screened by dot blot and ribonuclease protection assay to confirm that individual clones indeed represent differentially expressed genes. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as environmental pollutants.

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Management of Infections with Rapidly Growing Mycobacteria after Unexpected Complications of Skin and Subcutaneous Surgical Procedures

  • Lim, Jong-Min;Kim, Jong-Hwan;Yang, Ho-Jik
    • Archives of Plastic Surgery
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    • 제39권1호
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    • pp.18-24
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    • 2012
  • Background : Infection caused by rapidly growing mycobacteria (RGM) is not uncommon, and the prevalence of RGM infection has been increasing. Clinical diagnosis is difficult because there are no characteristic clinical features. There is also no standard antibiotic regimen for treating RGM infection. A small series of patients with RGM infections was studied to examine their treatments and outcomes. Methods : A total of 5 patients who had developed postoperative infections from January 2009 to December 2010 were retrospectively reviewed. Patients were initially screened using a mycobacteria rapid screening test (polymerase chain reaction [PCR]-reverse blot hybridization assay). To confirm mycobacterial infection, specimens were cultured for nontuberculous mycobacteria and analyzed by 16 S ribosomal RNA and rpoB gene PCR. Results : The patients were treated with intravenous antibiotics during hospitalization, and oral antibiotics were administered after discharge. The mean duration of follow-up was 9 months, and all patients were completely cured of infection with a regimen of a combination of antibiotics plus surgical treatment. Although none of the patients developed recurrence, there were complications at the site of infection, including hypertrophic scarring, pigmentation, and disfigurement. Conclusions : Combination antibiotic therapy plus drainage of surgical abscesses appeared to be effective for the RGM infections seen in our patients. Although neither the exact dosage nor a standardized regimen has been firmly established, we propose that our treatment can provide an option for the management of rapidly growing mycobacterial infection.

Diagnostic Performance of HPV E6/E7 mRNA and HPV DNA Assays for the Detection and Screening of Oncogenic Human Papillomavirus Infection among Woman with Cervical Lesions in China

  • Wang, Hye-young;Lee, Dongsup;Park, Sunyoung;Kim, Geehyuk;Kim, Sunghyun;Han, Lin;Yubo, Ren;Li, Yingxue;Park, Kwang Hwa;Lee, Hyeyoung
    • Asian Pacific Journal of Cancer Prevention
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    • 제16권17호
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    • pp.7633-7640
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    • 2015
  • Background: Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of cervical uterine cancer. Although HPV infections of the cervix do not always progress to cancer, 90% of cervical cancer cases have been found to be associated with high risk HPV (HR-HPV) infection. HPV DNA testing is widely used, along with Papanicolaou (Pap) testing, to screen for cervical abnormalities. However, there are no data on the prevalence of genotype-specific HPV infections assessed by measuring HPV E6/E7 mRNA in women representative of the Chinese population across a broad age range. Materials and Methods: In the present study, we compared the results with the CervicGen HPV RT-qDx assay, which detects 16 HR-HPV genotypes (Alpha-9: HPV 16, 31, 33, 35, 52, and 58; Alpha-7: HPV 18, 39, 45, 51, 59, and 68; and Alpha-5, 6: HPV 53, 56, 66, and 69), and the REBA HPV-ID assay, which detects 32 HPV genotypes based on the reverse blot hybridization assay (REBA) for the detection of oncogenic HPV infection according to cytological diagnosis. We also investigated the prevalence and genotype distribution of HPV infection with a total of 324 liquid-based cytology samples collected in western Shandong province, East China. Results: The overall HPV prevalences determined by HPV DNA and HPV E6/E7 mRNA assays in this study were 79.9% (259/324) and 55.6% (180/324), respectively. Although the positivity of HPV E6/E7 mRNA expression was significantly lower than HPV DNA positivity, the HPV E6/E7 mRNA assay showed greater specificity than the HPV DNA assay (88.6% vs. 48.1%) in normal cytology samples. The prevalence of Alpha-9 (HPV 16, 31, 33, 35, 52, and 58) HPV infection among these women accounted for up to 80.3% and 76.1% of the high-grade lesions detected in the HPV mRNA and DNA tests, respectively. The HR-HPV genotype distribution, based on HPV DNA and E6/E7 mRNA expression by age group in patients with cytologically confirmed lesions, was highest in women aged 40 to 49 years (35.9% for cytologically confirmed cases, Pearson correlation r value=0.993, p<0.001) for high-grade lesions. Among the oncogenic HR-HPV genotypes for all age groups, there was little difference in the distribution of HPV genotypes between the HPV DNA (HPV -16, 53, 18, 58, and 33) and HPV E6/E7 mRNA (HPV -16, 53, 33, 58, and 18) assays. HPV 16 was the most common HPV genotype among women with high-grade lesions. Conclusions: Our results suggest that the HPV E6/E7 mRNA assay can be a sensitive and specific tool for the screening and investigation of cervical cancer. Furthermore, it may provide useful information regarding the necessity for early cervical cancer screenings and the development of additional effective HPV vaccines, such as one for HPV 53 and 58. Additionally, gaining knowledge of HPV distribution may also inform us about ecological changes in HPV after the vaccination.