November 9, 2022
Dear Ryan Cuartero,
Greetings!
Thank you for your request dated Oct 19, 2022 11:36:09 AM under Executive Order No. 2 (s. 2016) on Freedom of Information in the Executive Branch.
Your Request
You asked for RT-PCR virus isolation and purification.
Response to Request
Your FOI request is approved. I enclose a copy of the information you requested.
1. Please explain the relevance and importance of virus isolation and purification.
-Virus isolation in the context of favorable selection and culture of the SARS-CoV-2 virus from a clinical specimen:
Virus Isolation is a method whereby virus targets in a clinical specimen (eg. Nasal swab in virus transport medium) are favorably cultured (purified) in cell lines, such that growth of other non-target pathogens are inhibited. Since pathogen/virus targets are expected to be in low concentrations in clinical specimens, which presents as a usual problem in the real of diagnostic testing, virus isolation via cell culture, whenever possible (high facility and technical requirements), is considered as an important tool in diagnostics and characterizing viruses. Virus isolation by cell culture, if successful, effectively increases the concentration of the virus target, which in turn increases confidence in the detection/identification result.
Virus isolation in the context of extracting the SARS-CoV-2 virus viral RNA from a clinical specimen:
Various technologies for viral RNA isolation and purification from clinical specimens (e.g. Nasal swab in virus transport medium) aim to separate viral RNA from the rest of the nucleic acids, proteins, cellular debris and other biomolecules that are present. This is carried out through various methods, but the currently more popular ones are the solid-phase extraction method (takes advantage of ionic properties of RNA in differential binding to a solid substrate for separation) and magnetic separation (magnetically separates RNA from the rest of the biomolecules present in the sample). Viral RNA isolation and purification is an important step prior to the detection of target SARS-CoV-2 diagnostic regions in the SARS-CoV-2 viral RNA via nucleic acid amplification tests (NAATS) such as SARS-CoV-2 Real-Time PCR.
2. What methods in Philippines do we use in isolating and purifying SARS-CoV2 virus?
-SARS-CoV-2 virus can be isolated by inoculation of Nasopharyngeal swab and Oropharyngeal swab specimens into Vero cells. Daily observation of the vero cells will be done using inverted microscope to monitor the appearance of Cytophatic Effect (CPE) or changes in the morphology of cell lines which is indicative of the presence of the virus, (Cell lines exhibiting epithelial morphology that was isolated from the kidney on an African Green Monkey, e.g. Vero E6, Vero/Hslam could be used).
Currently however, viral isolation by cell culture is not being performed, due to facility limitations.
For SARS-CoV-2 viral RNA isolation and purification from NPS and OPS, the methods available in the laboratory are manual extraction using spin columns and magnetic-bead based automated extraction.
Wurtz N, Penant G, Jardot P, Duclos N, La Scola B. Culture of SARS-CoV-2 in a panel of laboratory cell lines, permissivity, and differences in growth profile. Eur J Clin Microbiol Infect Dis. 2021 Mar;40(03);477-484. Doi: 10.1007/s10096-020-04106-0. Epub 2021 Jan2. PMID:33389257; PMCID: PMC7778494
3. What departments/Agencies/who do the isolation and purification of the SARS-CoV2 virus in Philippines?
-To our knowledge, there is no department or agency capable of viral isolation of SARC-CoV-2 in the Philippines as it requires a Biosafety Level 3 (BSL-3) laboratory. The risks of viral culture far outweigh the benefits when a more sensitive, specific and safer test such as RT-PCR is readily available.
Your right to request a review
If you are unhappy with this response to your FOI request, you may ask us to carry out an internal review of the response by writing to DOH Central Appeals and Review Committee, at
[email protected]. Your review request should explain why you are dissatisfied with this response, and should be made within 15 calendar days from the date when you received this letter. We will complete the review and tell you the result within 30 calendar days from the date when we receive your review request.
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Thank you.
Respectfully,
FOI Decision Maker
FOI Receiving Officer
