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mljgoums 2017, 11(1): 12-15 |
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Hajiahmadi N, Moradi A, Javid N, Tabarraei A. Limit of Detection of HEV RNA for Diagnosis of HEV Infection. mljgoums 2017; 11 (1) :12-15
URL: http://mlj.goums.ac.ir/article-1-925-en.html
URL: http://mlj.goums.ac.ir/article-1-925-en.html
1- Infectious Diseases Research Center
2- Infectious Diseases Research Center ,tabarraei@goums.ac.ir
2- Infectious Diseases Research Center ,
Abstract: (12833 Views)
ABSTRACT
Background and Objectives: Diagnosis of hepatitis E virus (HEV) infection could be missed in some cases if serological tests are used solely. Molecular characterization of HEV is essential for diagnosis of acute and chronic HEV infections, and evaluating the chronic HEV infection status in immunocompromised patients. The aim of this study was to prepare a suitable HEV positive control, determine the limit of detection (LOD) of HEV RNA for a specific molecular test, and evaluate the efficiency and precision of the test.
Methods: Genomic region of HEV NCBI reference sequence was constructed. LOD, intra-assay precision, and inter-assay precision were calculated to evaluate the efficiency and precision of the test. Then, tenfold serial dilutions of the HEV positive control were prepared. Real time PCR was performed three times for each dilution. Mean, standard deviation, and coefficient of variation of cycle thresholds obtained in three independent and simultaneous tests were calculated, and the results were analyzed.
Results: The LOD of this test was determined as 1.4×104 copy/ml or 42 copy/reaction or 14 copy/µl. Intra-assay precision and inter-assay precision for all assays were lower than 2.5% and 10%, respectively.
Conclusion: We propose that the real time PCR assay targeting the ORF2/3 overlapping conserved region is suitable for detection of a wide range of different HEV genotypes found in acute and chronic HEV infections. However, the precision of the test should be improved for detecting HEV RNA lower than 103 copy/ml.
Keywords: Hepatitis E virus, Limit of Detection, Real Time PCR.
Background and Objectives: Diagnosis of hepatitis E virus (HEV) infection could be missed in some cases if serological tests are used solely. Molecular characterization of HEV is essential for diagnosis of acute and chronic HEV infections, and evaluating the chronic HEV infection status in immunocompromised patients. The aim of this study was to prepare a suitable HEV positive control, determine the limit of detection (LOD) of HEV RNA for a specific molecular test, and evaluate the efficiency and precision of the test.
Methods: Genomic region of HEV NCBI reference sequence was constructed. LOD, intra-assay precision, and inter-assay precision were calculated to evaluate the efficiency and precision of the test. Then, tenfold serial dilutions of the HEV positive control were prepared. Real time PCR was performed three times for each dilution. Mean, standard deviation, and coefficient of variation of cycle thresholds obtained in three independent and simultaneous tests were calculated, and the results were analyzed.
Results: The LOD of this test was determined as 1.4×104 copy/ml or 42 copy/reaction or 14 copy/µl. Intra-assay precision and inter-assay precision for all assays were lower than 2.5% and 10%, respectively.
Conclusion: We propose that the real time PCR assay targeting the ORF2/3 overlapping conserved region is suitable for detection of a wide range of different HEV genotypes found in acute and chronic HEV infections. However, the precision of the test should be improved for detecting HEV RNA lower than 103 copy/ml.
Keywords: Hepatitis E virus, Limit of Detection, Real Time PCR.
Research Article: Original Paper |
Received: 2017/01/28 | Accepted: 2017/01/28 | Published: 2017/01/28 | ePublished: 2017/01/28
Received: 2017/01/28 | Accepted: 2017/01/28 | Published: 2017/01/28 | ePublished: 2017/01/28
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