Can qPCR differentiate Vax Spike from C19 Spike

Several pathologists have asked us how to differentiate Vax Spike from COVID Spike.

This is important in order to differentiate Long Vax from Long COVID and its traditionally done by looking for nucleocapsid protein with ELISA or other protein based assays. The vaccine doesn’t present any nucleocapsid sequence so only those infected by the virus should have both spike and nucleocapsid.

The Virus spike and Vax spike only different by two prolines and no tests are available that can differentiate these subtle changes at the protein level. Mass Spec in theory can.

Due to the codon optimizations of the vaccines, the DNA sequences for these two versions of spike are substantially different. Pfizer is only 72% identical to the Wuhan1 spike DNA sequence.

BLAST alignment of the Pfizer Spike sequence to Wuhan-1

This makes it very easy to differentiate the Vax spike residual DNA or RNA from the virus.

To confirm this, we ordered 2 gBlocks (synthesized dsDNA from IDT) that represent the vax Spike and the virus spike. This region of the Vax spike is identical between Modern, Janssen and Pfizer. We then tested if our primers have any activity on the Pfizer gBlock versus the Wuhan1 gBlock across a serial dilution in duplicate.

This started at 1ng/ul with 10 fold dilutions.

Signal is seen with the Pfizer gBlock (blue). This is not surprising given the many labs that have now reproduced this work on the vaccines (Lee et al, Buckhaults et al).

What was surprising was to find zero cross reactivity with the Wuhan-1 sequence for 40 cycles. I was expecting a 10-20 CT offset but instead the primers show ZERO reactivity with the Virus spike gBlock.

This is a very favorable outcome for using these assays as tool to track Long Vax and Long Covid biopsies. Conventional C19 qPCR kits can be used to look for the virus and these vaccine targeted kits can be used to assess tissues, blood, semen, saliva, monocytes etc. for evidence of residual DNA or mRNA from the vaccines.

We would recommend testing for VaxSpike (FAM), VaxOri(HEX) VaxSV40enhancer (TXRED) and RNAaseP(CY5). This is a 4 color assay where the SV40 Enhancer provides the most sensitivity due to having 2X the copy number in the vaccine. The SV40Enhancer is also only present in Pfizer so SV40 target failure with Spike positive qPCR would indicate Moderna vaccination and not Pfizer vaccination. The RNaseP assay is direct from IDT and used in C19 testing. This confirms you have DNA and RNA extracted properly from the tissue (internal control for qPCR). The VaxOri is present in both Moderna and Pfizer but not Janssen.

A logic matrix for deducing which vaccine is in the tissue is shown in table 1. This gets more complicated as they mix and match vaccines in the same patient but presumably the most recently vaccine administered will dominate the signal.

Table 1. Logic table to deduce which vaccine is present in a given patient.

If you would like more information regarding these assays, contact us at info@medicinalgenomics.com