Comparison of real-time reverse transcriptase–polymerase chain reaction and nested or commercial reverse transcriptase–polymerase chain reaction for the detection of hepatitis E virus particle in human serum
Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus Herpesvirus of the family Herpesviridae. Recently, HEVs were identified from sev...
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| Veröffentlicht in: | Diagnostic microbiology and infectious disease Jg. 56; H. 3; S. 269 - 274 |
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| Abstract | Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus
Herpesvirus of the family Herpesviridae. Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase–polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 × 10
1 copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR. |
|---|---|
| AbstractList | Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus
Herpesvirus of the family Herpesviridae. Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase–polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 × 10
1 copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR. Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single- stranded positive RNA virus and has been classified in the genus Herpesvirus of the family Herpesviridae. Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase-polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 x 10 copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR. Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus Herpevirus [corrected] of the [corrected] Herpeviridae [corrected] Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase-polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 x 10(1) copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR.Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus Herpevirus [corrected] of the [corrected] Herpeviridae [corrected] Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase-polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 x 10(1) copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR. Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus Herpevirus [corrected] of the [corrected] Herpeviridae [corrected] Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase-polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 x 10(1) copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR. |
| Author | Rayamajhi, Nabin Ahn, Jeong-min Sang Yoo, Han Gyun Kang, Sang |
| Author_xml | – sequence: 1 givenname: Jeong-min surname: Ahn fullname: Ahn, Jeong-min – sequence: 2 givenname: Nabin surname: Rayamajhi fullname: Rayamajhi, Nabin – sequence: 3 givenname: Sang surname: Gyun Kang fullname: Gyun Kang, Sang – sequence: 4 givenname: Han surname: Sang Yoo fullname: Sang Yoo, Han email: yoohs@snu.ac.kr |
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| CitedBy_id | crossref_primary_10_2903_j_efsa_2017_4886 crossref_primary_10_1002_jmv_21116 crossref_primary_10_1016_j_patbio_2010_04_004 crossref_primary_10_1128_JCM_01626_10 crossref_primary_10_1128_JCM_03118_13 crossref_primary_10_1007_s00103_008_0423_y crossref_primary_10_1016_j_eururo_2008_04_059 crossref_primary_10_1016_j_urology_2011_09_006 crossref_primary_10_1371_journal_pone_0146906 crossref_primary_10_17221_1999_VETMED crossref_primary_10_3390_microorganisms10020428 crossref_primary_10_1016_j_jviromet_2007_07_014 crossref_primary_10_1016_j_jviromet_2014_05_026 crossref_primary_10_1159_000113057 crossref_primary_10_1016_j_jviromet_2011_05_001 crossref_primary_10_1128_JCM_05942_11 crossref_primary_10_1159_000357098 crossref_primary_10_1111_j_1365_2672_2008_04104_x crossref_primary_10_1002_jmv_21040 crossref_primary_10_1007_s00705_010_0679_z crossref_primary_10_3390_foods13030467 crossref_primary_10_1128_spectrum_01912_21 |
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| Keywords | Real-time RT-PCR HEV Human RNA-directed DNA polymerase Microbiology Enzyme Transferases Real time Hepatitis E virus Virus Infection Polymerase chain reaction Calicivirus Nucleotidyltransferases Caliciviridae Serum Detection Reverse transcription polymerase chain reaction |
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| SubjectTerms | Biological and medical sciences Fundamental and applied biological sciences. Psychology Hepatitis Antibodies - blood Hepatitis E - diagnosis Hepatitis E virus Hepatitis E virus - classification Hepatitis E virus - genetics Hepatitis E virus - immunology Hepatitis E virus - isolation & purification Herpesvirus HEV Humans Infectious diseases Medical sciences Microbiology Miscellaneous Real-time RT-PCR Reverse Transcriptase Polymerase Chain Reaction - methods RNA, Viral - blood Sensitivity and Specificity Virology |
| Title | Comparison of real-time reverse transcriptase–polymerase chain reaction and nested or commercial reverse transcriptase–polymerase chain reaction for the detection of hepatitis E virus particle in human serum |
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