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العنوان
Detection of Hepatitis C Virus genotype-4 using nucleic acid aptamers /
المؤلف
Gouda, Abdullah Elsayed Mohamed.
هيئة الاعداد
باحث / Abdullah Elsayed Mohamed Gouda
مشرف / Amina Mohamed Medhat
مشرف / Mohamed Abbas Shemis
مناقش / Soha Mohamed Hamdy,
تاريخ النشر
2019.
عدد الصفحات
239 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
Biochemistry
تاريخ الإجازة
1/1/2019
مكان الإجازة
جامعة عين شمس - كلية العلوم - قسم الكيمياء الحيوية
الفهرس
Only 14 pages are availabe for public view

from 239

from 239

Abstract

5. Summary
Hepatitis C virus (HCV) infection is a major contributing agent to mortality in the whole world. Early investigation and curative treatment of HCV can reduce the risk of liver-related mortality and serve to stop transmission of new infections. Various Egyptian studies described HCV as a major public health burden,with the highest prevalence rate in the world. Different HCV rapid tests and screening Enzyme-Linked Immunosorbent Assays (ELISAs) have been available for detection of HCV. These assays lack the higher sensitivity, specificity and cannot detect the early infection, immuno-suppressed patients succeeding transplantation and the immunocompromised HIV-infected pateints. Hence, both the positive and negative samples obtained using these assays require to be confirmed using the qRT-PCR method which is more expensive, time consuming and prone to contamination technique.
The present study aimed to design a sensitive aptasensor composed of thiol modified nucleic acid aptamer and gold nanoparticles (AuNPs) for the early detection of HCV. HCV Core recombinant protein was expressed, purified and refolded, then the serological reactivity of the purified recombinant Core protein was tested using ELISA assay. The results showed a promising, more effective and high-level yield of expressed protein. The purified HCV Core protein was first used to develop a modified ELISA assay. The results of the modified ELISA assay were highly sensitive, more specific and reproducible compared to commercial ELISA kit. Together with the purified HCV Core recombinant antigen, the HCV Core aptasensor was utilized to develop the highly sensitive, more specific, cost-effective and rapid HCV Core aptamer colorimetric assay. A total of 135 archived serum samples divided into two groups were enrolled in this study: 75 archived serum samples collected from patients with HCV infection along with 60 HCV negative control samples. All samples were collected from the Biochemistry and Molecular Biology laboratory, Theodor Bilharz Research Institute (TBRI). A total of 69 (92%) of the 75 positive samples were genotype-4 and selected for re-testing by the HCV Core aptamer colorimetric assay, in-house anti-HCV Core assay and the commercial ELISA kit along with 60 negative control samples. These positive samples were of different viral loads ranging from 255 IU to 6,934,590 IU.
All the 69 HCV psitive samples and 60 HCV negative samples were detected by the HCV Core aptamer colorimetric assay with sensitivity and specificity of 100%, 68 samples were confirmed by the in-house anti-HCV Core assay (~98.55 sensitivity) and 66 samples were confirmed by the commercial ELISA kit (~95.65 sensitivity). The correlation analysis between the two developed assays and the golden reference qRT-PCR technique showed higher correlation of HCV Core aptamer colorimetric assay to the qRT-PCR, where as the in-house anti-HCV core assay (ELISA-based assay) was of lower correlation.
The HCV Core aptamer colorimetric assay exceeds other methods as a simple, easy to perform, rapid, more sensitive and cost-effective and early detection technique.
Conclusion and recommendations
High-level of the purified recombinant HCV genotype-4 Core antigen was obtained and used as capturing antigen for detection of HCV antibodies in an ELISA assay with high sensitivity, specificity and enhanced reproducibility compared to the commercial ELISA kit. This recombinant protein was also utilized as a reference standard for detection of HCV Core antigen using the highly sensitive and more specific HCV Core aptamer colorimetric assay. HCV Core aptamer colorimetric assay differentiate between the chronic and resolved HCV infection through its real count of the HCV core antigen. This promising assay can be recommended for the in-house screening and quantification of HCV infection.