@article{10.11648/j.ajim.20210901.11,
  author = {Khaldoun Al-Romaih and Ibtihaj Alsharif and Razan Bakheet and Lina Mahmoud and Najla Alharbi and Sara Bin Judia and Layla Alharbi and Abdulaziz Alzayed and Amjad Jabaan and Jawahar Alotaibi and Alaiya Ayodele and Zakiya Shinwari and Hani Alhadrami and Ahmed Albarrag and Esam Azhar and Haya Al-Saud and Tahani Alrahbini and Maha Al-Mozaini},
  title = {Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing},
  journal = {American Journal of Internal Medicine},
  volume = {9},
  number = {1},
  pages = {1-10},
  doi = {10.11648/j.ajim.20210901.11},
  url = {https://doi.org/10.11648/j.ajim.20210901.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajim.20210901.11},
  abstract = {Background: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. Methods: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMaxTM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). Results: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. Conclusion: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing.},
 year = {2021}
}

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TY  - JOUR
T1  - Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing
AU  - Khaldoun Al-Romaih
AU  - Ibtihaj Alsharif
AU  - Razan Bakheet
AU  - Lina Mahmoud
AU  - Najla Alharbi
AU  - Sara Bin Judia
AU  - Layla Alharbi
AU  - Abdulaziz Alzayed
AU  - Amjad Jabaan
AU  - Jawahar Alotaibi
AU  - Alaiya Ayodele
AU  - Zakiya Shinwari
AU  - Hani Alhadrami
AU  - Ahmed Albarrag
AU  - Esam Azhar
AU  - Haya Al-Saud
AU  - Tahani Alrahbini
AU  - Maha Al-Mozaini
Y1  - 2021/01/04
PY  - 2021
N1  - https://doi.org/10.11648/j.ajim.20210901.11
DO  - 10.11648/j.ajim.20210901.11
T2  - American Journal of Internal Medicine
JF  - American Journal of Internal Medicine
JO  - American Journal of Internal Medicine
SP  - 1
EP  - 10
PB  - Science Publishing Group
SN  - 2330-4324
UR  - https://doi.org/10.11648/j.ajim.20210901.11
AB  - Background: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. Methods: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMaxTM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). Results: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. Conclusion: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing.
VL  - 9
IS  - 1
ER  - 

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