Hello Annadurai,

Greetings!

I hope that you are familiar with purposes of VTM in sample collection.

Let's discuss about the composition of VTM, which mainly consists of

1) 2 % FBS to preserve the sample

2) 100 µg /mL Gentamicin to suppress the growth of contaminating bacteria

3) 0.5 µg /mL Fungizone to suppress the growth of contaminating fungi

4) Hanks Balanced Salt Solution (HBSS) 1X with calcium and magnesium ions as a buffer

We can easily understand that these compounds do not interfere RNA isolation from swap samples, since the step containing few centrifugation steps. Moreover, the VTM samples may be centrifuged and remove supernatant; only pellet may be used for RNA isolation (I am not sure about this step!).

My straight answers are

1. Does a 4-fold reduction of the VTM from 3ml to 0.75ml improve the viral load in the sample?

No, a 4-fold reduction of the VTM from 3ml to 0.75ml does not improve the viral load in the sample? because prior to RNA isolation, VTM may be subjected to high speed centrifugation (usually viral RNA isolation procedure starts with this step).

2. Is it logical that reducing the volume of VTM may reduce the false-negative results of COVID-19 testing by increase the viral load in the sample?

As we are familiar with purposes of VTM, a slight change in volume does not affect the test results. False negative is associated with viral load of actual samples. However, we should consider principle of qualitative real-time PCR here.

3. What is the rationale behind the fixed volume of VTM as 3 ml? Is it fixed arbitrarily or due to any technical reasons?

This is may be due to preserve the sample, 3 mL of VTM is adequate for well preservation of samples.

Please share your opinions with me, kindly note that my answers are based on only theoretical knowledge.

Thanks!

References

1) https://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport- Medium.pdf

2) https://microbeonline.com/viral-transport-media-vtm/

3) https://www.bd.com/en-us/offerings/capabilities/specimen-collection/swab-based-specimen-collection/bd-universal-viral-transport-system

Hai Badrinath,

Thank you for your clarifications.

Actually, 3ml of sample is not processed to obtain a RNA pellet. The RNA extraction starts from 200µl of this sample and Pellet RNA is formed only from this 200µl sample.

Note: In some laboratories, 3ml sample may be centrifuged at low RPM for sendimenting heavy debris. After sendimentation, 200µl sample is taken and processed for RNA extraction.

Hi Annadurai,

Thanks for reply,

Please note that each RNA extraction kit has some modifications in the protocol for minimal RNA extraction. For example, poly (A)RNA or spin filter membrane is used to attract more RNA in a sample. As I early mentioned, minimum viral copy number is considered in qualitative real-time PCR to detect virus presence in a sample (negative or positive). In this case, 200 uL is enough to detect minimum viral RNA copies.

Moreover, each kit has its own detection limit, based on this limit, manufacturer has developed cutoff value for CT (e.g 50 copies = CT 35).

In my opinion, the purpose of 3 mL of VTM is for transportation and preservation of samples.

For more clarifications, please refer to references.

References