• Title/Summary/Keyword: nested-PCR

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Detection and genetic analysis of zoonotic hepatitis E virus, rotavirus, and sapovirus in pigs

  • Lyoo, Eu Lim;Park, Byung-Joo;Ahn, Hee-Seop;Han, Sang-Hoon;Go, Hyeon-Jeong;Kim, Dong-Hwi;Lee, Joong-Bok;Park, Seung-Yong;Song, Chang-Seon;Lee, Sang-Won;Choi, In-Soo
    • Korean Journal of Veterinary Research
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    • v.60 no.2
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    • pp.61-68
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    • 2020
  • The zoonotic transmission of viral diseases to humans is a serious public health concern. Pigs are frequently a major reservoir for several zoonotic viral diseases. Therefore, periodic surveillance is needed to determine the infection rates of zoonotic diseases in domestic pigs. Hepatitis E virus (HEV), rotavirus, sapovirus (SaV), and norovirus (NoV) are potential zoonotic viruses. In this study, 296 fecal samples were collected from weaned piglets and growing pigs in 13 swine farms, and the viral RNA was extracted. Partial viral genomes were amplified by reverse transcription-polymerase chain reaction (PCR) or nested-PCR using virus-specific primer sets under different PCR conditions. HEV-3, rotavirus A, and SaV genogoup 3 were detected from 11.5, 2.7, and 3.0% of the samples, respectively. On the other hand, NoV was not detected in any of the samples. Genetic analysis indicated that the nucleotide sequences of swine HEV-3 and rotavirus A detected in this study were closely related to those of human isolates. However, swine SaV was distant from the human strains. These results suggest that HEV-3 and rotavirus A can be transmitted from pigs to humans. Therefore, strict preventive measures should be implemented by workers in the swine industry to prevent infections with HEV-3 and rotavirus A excreted from pigs.

Prevalence of Human Papillomavirus Types and Phylogenetic Analysis of HPV-16 L1 Variants from Southern India

  • Kabekkodu, Shama Prasada;Bhat, Samatha;Pandey, Deeksha;Varghese, Vinay Koshy;Shukla, Vaibhav;Ghosh, Supriti;Kushtagi, Pralhad;Bhat, Parvati;Gopinath, Puthiya Mundayat;Satyamoorthy, Kapaettu
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.5
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    • pp.2073-2080
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    • 2015
  • Background: The human papillomavirus (HPV) and its variants show wide geographical distribution and have been reported to cause cervical lesions. With cervical neoplasia as the leading cancer in Indian women, the aim of the present study was to evaluate the multiple infection HPV type distribution and variant genotypes in cervical samples from the coastal Karnataka region, India. Materials and Methods: A total of 212 samples were screened by nested polymerase chain reaction using PGMY9/11 and GP5+/6+ primers. HPV positive samples were sequenced to identify the types and a phylogenetic tree was constructed using the neighbor-joining method. Results: Sequence analysis identified a total of 14 HPV types distributed in 20%, 73.3% and 82.5% of non-malignant, pre-malignant [low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL)] and cervical cancer samples. The distribution of high risk HPV in cancer samples was HPV 16, 76.4%, HPV18, 11.7%, HPV81, 2.9%, HPV31, 1.4%, HPV35, 1.4% and HPV 45, 1.4%. Multiple infections were observed in 11.8% of tumor samples with HPV 16 contributing to 62.5% of cases. In non-malignant samples, 20% of HPV positive samples were detected with HPV16, 82.3%, HPV33, 5.8% and HPV58, 5.8% and very low incidence of multiple infections. Comparative phylogenetic analysis of HPV variants identified 9 HPV sequences as new papillomavirus species, predominantly classified as European lineage type. Conclusions: The findings for HPV infections associated with progression of cervical cancer in coastal Karnataka region and HPV variant analysis provide baseline data for prevention and HPV vaccination programs.

How Reliable is Sputum PCR Test in the Diagnosis of Pulmonary Tuberculosis When Sputum Smear is Negative? (객담 결핵균 도말검사가 음성일때 중합효소연쇄반응검사와 진단적 신뢰도에 관한 연구)

  • Baek, Seung-Hoon;Lee, Jae-Myung;Kang, Min-Jong;Son, Jee-Woong;Lee, Seung-Joon;Kim, Dong-Gyu;Lee, Myung-Goo;Hyun, In-Gyu;Jung, Ki-Suck;Lee, Kyung-Wha;Joe, Hyun-Chan
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.2
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    • pp.222-228
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    • 2001
  • Backgrounds : Recent technological developments have introduced a new method to identifying M. tuberculosis complex DNA in clinical samples directly. The direct amplification test (DAT) is approved for identifying M. tuberculosis complex in respiratory specimens that are smear-positive for acid-fast bacilli (AFB). When there is a discrepancy between the AFB smear and DAT, no information on their clinical utility is currently available. In this study, the diagnostic reliability of DAT was investigated in suspected pulmonary tuberculosis patients whose sputum AFB smear was negative. Methods : From June 1, 1998 through May 30, 1999, 909 patients with presumed active pulmonary tuberculosis were enrolled. A sputum AFB stain, culture, DAT and/or biopsy were performed. Using the criteria of clinical tuberculosis or confirmed tuberculosis, the positive predictive value of DAT in diagnosing pulmonary tuberculosis was investigated. Results : The positive predictive value of DAT was 82.1% by the clinically active tuberculosis criteria. However, it decreased to 61.5% when diagnosis was restricted to only to culture positive or biopsy proven cases. The false positive rate of DAT was 18.0%. Conclusion : The DAT is a valuable diagnostic method in suspected patients whose sputum AFB is was negative.

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Sequence and Phylogenetic Analysis of V3 Region of Human Immunodeficiency Virus Type 1 Strains Isolated from Korean Patients (한국인 인면역결핍 바이러스의 V3 Loop 염기서열 분석 및 계통발생학적 분석)

  • Kim, Young-Bong;Cho, Young-Keol;Lee, Hee-Jung;Chung, Koo-Hun;Kim, Jung-Woo;Kim, Yoo-Kyum;Yang, Jai-Myung
    • The Journal of Korean Society of Virology
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    • v.26 no.2
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    • pp.251-258
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    • 1996
  • The V3 loop, a hypervariable domain of envelope glycoprotein, has an essential role in viral infectivity and has a major epitope for type-specific neutralizing antibody. In order to investigate genetic diversity of V3 region of gp120 of human immunodeficiency virus type 1 (HIV-1) isolated from Korean patients, DNA sequences encoding the C2 to V3 region were amplified by nested polymerase chain reaction (PCR) from uncultured peripheral blood mononuclear cells obtained from 15 HIV-1 seropositive patients and nucleotide sequences were determined. All nucleotide sequences from fifteen patients were compared with 8 distinctive subtypes (A-H) and another subtype O. Phylogenetic analysis was carried out with PHYLIP ver 3.5 (Dnapars) program. Of the 15 isolates, 14 HIV-1 subjects were clustered with subtype B, while one was clustered with subtype C. Intra-subtype B distance at the nucleotide and deduced amino acid level were maximum 17.7% and 37.0%, respectively. Intra-patient distance at the nucleotide and deduced amino acid level were maximum 7.3% and 17.8%, respectively. Analysis of the nucleotide sequences revealed that Korean types have relatively well conserved sequences. These findings could be useful for assessing the source of infection and developing an AIDS vaccine.

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Korean red ginseng attenuates HIV-1 vivo; High frequency of grossly deleted nef genes in HIV-1 infected long-term slow progressors treated with Korean red ginseng - Running title: Grossly deleted nef genes in slow progressors -

  • Cho, Young-K.;Lim, Ji-Y.;Jung, You-S.;Oh, Sun-K.;Lee, Hee-J.;Sung, Heung-Sup
    • Proceedings of the Ginseng society Conference
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    • 2006.05a
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    • pp.40-51
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    • 2006
  • To investigate the association between Korean red ginseng (KRG) intake in HIV-1 infected patients and occurrence of grossly deleted nef genes ($g{\Delta}nef$), we characterized nef genes in 10 long-term slow progressors (LTSP) infected with HIV-1 subtype B and 34 control patients. LTSP was defined whose the annual decrease in CD4 T cells was less than $20/{\mu}l$ over 10 years in the absence of antiretroviral therapy. They were treated with KRG for a prolonged period. Nef genes were amplified from peripheral blood mononuclear cells (PBMC) using nested PCR and products were sequenced directly. Patient CD4 T cell counts decreased from $444{\pm}207/{\mu}l$ to $294{\pm}177/{\mu}l$ over $136{\pm}23$ months of KRG intake. This corresponds to an annual decrease in the level of CD4 T cells of $13.3/{\mu}l$. A total of 479 nef genes were amplified from 137 PBMC samples. Nine out of the 10 patients, 47 (34.3%) out of the 137 samples, and 92 out of the 479 genes revealed $g{\Delta}nef$. The deletion extended outside the nef gene in 25 $g{\Delta}nef$ obtained from 6 patients. The proportion of samples with $g{\Delta}nef$ (34.3%) was significantly higher than 4.8% in control patients (P<0.001). In addition, it significantly increased as the duration of KRG intake prolongs (P<0.01). These data suggest the possibility that occurrence of $g{\Delta}nef$ might be associated with long-term intake of KRG.

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Comparison of Microbial Diversity of Korean Commercial Makgeolli Showing High ${\beta}$-Glucan Content and High Antihypertensive Activity, Respectively

  • Min, Jin-Hong;Kim, Young-Hun;Kim, Jae-Ho;Choi, Shin-Yang;Lee, Jong-Soo;Kim, Ha-Kun
    • Mycobiology
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    • v.40 no.2
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    • pp.138-141
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    • 2012
  • We measured physiological functionalities, including antihypertensive angiotensin I-converting enzyme inhibitory activity and immun-stimulating ${\beta}$-glucan content for sixty kinds of Makgeolli that is commercially available from the market. As a result, we selected R-12 commercial raw Makgeolli, with a high content of immuno-stimulating ${\beta}$-glucan, and R-14 commercial raw Makgeolli, exhibiting high antihypertensive activity. Due to the similarities in their overall physicochemical properties and raw materials used for fermentation, we compared the microbial flora in order to investigate the reason for the differences in their functionalities. Nested PCR and denaturing gradient gel electrophoresis for yeasts and bacteria were performed for analysis of microbial diversity of two different kinds of Makgeolli (i.e., R-12, R-14), which showed immuno-stimulating ${\beta}$-glucan content and exhibited a very high level of antihypertensive activity, respectively. Analysis of the 18S rDNA amplicon revealed a major presence of the yeast strain Pichia burtonii in every Makgeolli sample. Analysis of the 16S rDNA amplicon revealed a predominance of lactic acid bacteria, and the most frequent lactic acid bacteria were Lactobacillus ingluviei, L. fermentum, and L. harbinensis, and Lactobacillus sp. Among these, L. harbinensis was detected only in R-12 and L. ingluviei was found only in R-14. Different functionalities from the individual commercially available Makgeolli may be attributed to actions of different microbial flora during fermentation.

Detection of Helicobacter Pylori in Saliva of Korean Infant (한국인 소아의 타액에서 Helicobacter pylori의 검출)

  • Ryu, Ji-Won;Lee, Jang-Keun;Lee, Yong-Su;Yoon, Chang-Lyuk;Ahn, Jong-Mo
    • Journal of Oral Medicine and Pain
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    • v.33 no.1
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    • pp.9-14
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    • 2008
  • Helicobacter pylori(H. pylori) has been associated with the cause of peptic ulcer and gastric cancer. H. pylori infection occur mostly during childhood and increase by aging. In route of transmission, Oral cavity does important role. So we employed this study to elucidate route of transmission by detection of H. pylori in infant saliva. We investigated 20 infants aged below 10 years and 20 teens aged below 20 years as study group and 71 adults aged 20 and over years as control group. H. pylori DNA was isolated from 5(25%) infants aged below 10 years, 6(30%) teens and 17(23.9%) adults by nested polymerase chain reaction(n-PCR). There was no statistically significant difference(P>0.05). The obtained results suggest that H. pylori infection is relatively common in saliva of Korean infant and oral cavity may be reservoir of H. pylori.

Detection of Helicobacter pylori in Saliva of Patient with Oral Lichen Planus (구강 편평태선 환자의 타액에서 Helicobacter pylori의 검출)

  • Ryu, Ji-Won;Kang, Seung-Woo;Yoon, Chang-Lyuk;Ahn, Jong-Mo
    • Journal of Oral Medicine and Pain
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    • v.33 no.3
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    • pp.241-246
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    • 2008
  • Lichen planus is a common, chronic inflammatory disease of the skin and mucous membrane for which no precise causes have been confirmed. But it is often connected with infections. Helicobacter pylori(H. pylori) among various bacteria has been associated with the cause of gastritis, peptic ulcer and gastric cancer. Considering the similarities of histological features between gastric ulcer and oral ulcers, it is resonable to assume that H. pylori might also be involved in the development oral mucosal ulceration. So we employed this study to investigate the possible involvement of H. pylori in the aetiology of erosive oral lichen planus. We analyzed detection rate of H. pylori in saliva of patients with erosive oral lichen planus by nested PCR. As a result, it revealed a significant difference statistically by showing positivity in 16 to 21(76.2%) saliva samples of patients group and in 11 of 44(25%) saliva samples of control group(P>0.001). We were able to suppose that H. pylori in saliva can be related to cause of erosive oral lichen planus.

Congenital Malaria in Newborns Selected for Low Birth-Weight, Anemia, and Other Possible Symptoms in Maumere, Indonesia

  • Fitri, Loeki Enggar;Jahja, Natalia Erica;Huwae, Irene Ratridewi;Nara, Mario B.;Berens-Riha, Nicole
    • Parasites, Hosts and Diseases
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    • v.52 no.6
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    • pp.639-644
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    • 2014
  • Congenital malaria is assumed to be a risk factor for infant morbidity and mortality in endemic areas like Maumere, Indonesia. Infected infants are susceptible to its impact such as premature labor, low birth weight, anemia, and other unspecified symptoms. The aim of this study was to investigate the prevalence of congenital malaria and the influence of mother-infant paired parasite densities on the clinical outcome of the newborns at TC Hillers Hospital, Maumere. An analytical cross sectional study was carried out in newborns which showed criteria associated with congenital malaria. A thick and thin blood smear confirmed by nested PCR was performed in both mothers and infants. The association of congenital malaria with the newborn's health status was then assessed. From 112 mother-infant pairs included in this study, 92 were evaluated further. Thirty-nine infants (42.4%) were found to be infected and half of them were asymptomatic. Infected newborns had a 4.7 times higher risk in developing anemia compared to uninfected newborns (95% CI, 1.3-17.1). The hemoglobin level, erythrocyte amount, and hematocrit level were affected by the infants' parasite densities (P<0.05). Focusing on newborns at risk of congenital malaria, the prevalence is almost 3 times higher than in an unselected collective. Low birth weight, anemia, and pre-term birth were the most common features. Anemia seems to be significantly influenced by infant parasite densities but not by maternal parasitemia.

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

  • Congpuong, Kanungnit;Hoonchaiyapoom, Thirasak;Inorn, Kornnarin
    • Parasites, Hosts and Diseases
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    • v.52 no.6
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    • pp.631-637
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    • 2014
  • Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., $K1_{270-290}$, $3D7_{610-630}$, $G_{650-690}$, while 2 variants, $K1_{150-170}$, and $3D7_{670-690}$ were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.