PCR007: Running RT-qPCR on undiluted/unconcentrated COVID wastewater RNA samples (open)

This is a copy of the original notebook created by Trevor Ho and Nahuel Moreno in Nick Gilbert group (https://chromatinlab.org/) for the COVID Wastewater Scotland project (https://biordm.github.io/COVID-Wastewater-Scotland/)

We need an answer on whether RT-qPCR can detect SARS-CoV-2 from wastewater.

Nick said the wastewater samples we got contain SARS-CoV-2 RNA.

If I look back at January, the majority of mutants should be alpha and WT, globally, and it should be mostly alpha variant in the UK.

Source: https://nextstrain.org/ncov/gisaid/global?dmax=2021-02-23&dmin=2020-12-24&gmin=15

This is global statistics of overall what proportions are there.

What this shows, on 6th Feb (wastewater sample collection time point)

Alpha: 19%

Beta: 11%

Gamma: 3%

Clade 20 (presumably WT): 60%

Based on this observation, it should be expected that I should be able to amplify some signal from the wastewater with predominantly WT strains.

We should be able to get sufficient amplification from some wastewater samples. Just need to find it out now.

Planning:

duplicates for each sample

could try NEB mix this time (and make use of signal normalization by ROX), or put in ROX inside the Takara Kit

Want to try as many wastewater samples as possible

From PCR004_R2, it was known that the extracted positive control works. So I must use it for check how things work

After thinking more about it, instead of putting a separate well for the human / coronavirus checking controls, it is better to multiplex some of probes together so this leaves more wells for other things and duplicates. Designated one mix as MultiPlex Mix (MPM)

Samples:

Many of the samples are leftover samples from ​Inaccessible Entry Run1.

Given reanalysis of data from ​Inaccessible Entry , also tried for the first time to incorporate ROX passive reference into the reaction. ROX will share a channel with TXRD, so don't know how it will work, but let's see how it goes.

According to https://www.takarabio.com/documents/User%20Manual/RR600A_RR600S_e.v2008Da.pdf,

dye is 50 X, so 0.2 μL per 10 μL reaction.

Ran RT-qPCR using conventional program.

Ran RT-qPCR overnight.

Result analysis for PCR007_R1:

N gene probably is sometimes reliable in checking for SARS-CoV-2 RNA in wastewater RNA samples. Not sure contamination in NTC or what.

E gene had difficulty giving any signals from wastewater RNA samples, also one contamination in NTC

Could not amplify anything from S gene using any of the primer probe sets, even if they work very well in EPC samples

Primer and probes of beta actin gave strong signal in EPC, contamination? But otherwise, beta-actin RNA readily detected in wastewater RNA samples

RPP30 gave nothing in any of the samples, doesn't look like something promising to track the samples

We will next try (again) to amplify some covid material from the SEPA samples. As the first run failed, we have a few options:

Increase the concentration of the primers for increasing the sensitivity.

Increase the time of RT reaction.

We will use the SEPA samples undiluted, and will use all of the RNA we have in them. If we don't have enough for all the reactions we will have to pool them again.

Plate setup:

Mastermixes:

A selection of the samples was run on a 2% agarose TBE gel.

Gel setup:

*By 5 µL of sample load volume, the actual volume loaded was 6 µL, since most likely 2 µL of 6X loading dye was mixed with the PCR product.

The gel was ran at 75 V for around 2 hours. Post-stained gel in 400 mL water + 10 µL EtBr for > 30 mins.

Result:

Turned up the diaphragm:

The resultes obtained with the Takara Primescript III kit are confusing. SEPA routinely use the NEB Luna kit so we will run a smaller scale experiment using this kit. This is partly due to the unavailability of the kit. We might have to resort to NEB Luna + UDG.

We will use standard kit specifications and we will not double the concentration of primers.

Plate setup:

Mastermix:

We run a gel with some of the samples to check for elongation. β-actin EPC showed strange amplification in the qPCR in wells E5 and E6 so we will run all the samples in the gel to see how they look.

Very bad gel image, I should have checked it before throwing away the gel.

All of the EPC samples give clear signals and amplification (orange dots).

FAM probes, except the N_gene probe, have a marked basal signal which is seen in the NTC wells. We have to look into this.

NTC did not display amplification for E_gene, N_gene and β-actin, which suggests there has been succesful amplification of the genes. As expected, EPC did not display β-actin amplification, but there might be contamination or unspecific amplification, as two wells showed bands in the gel, and some late amplification has been seen in the qPCR.

The gel shows strong bands in many of the samples, including NTC wells, which is puzzling.

​Trevor Y. H. Ho: I wish to try and see if we could at least get some consistent amplification from the wastewater. Any wastewater samples processed so far have difficulty producing consistent results.

Have been looking more into how SEPA process their wastewater and what primers and probes they used.

Points noted:

Viral RNA was concentrated in untreated sludge. Quote from page 7: "SARS-CoV-2 RNA was detected at higher levels in primary sludge than influent"

Also checked the paper from Bangor which was referenced in CREW's report. Basically, their were using the same set of primers and probes as we are.

Given so, there really isn't any reason why we weren't able to detect something from the wastewater.

The most plausible reason I could imagine then, is that their RNA samples arrived heavily degraded, either because they have been sitting in the freezer for too long, or they got degraded during the transportation process.

Looking at their recipe there are a couple of things that could be drawn:

They run their reactions in triplicates

They run a larger volume

They use a larger proportion of template volume, 1/4 of total reaction volume, while we only used 1/10

They use BSA

Regarding BSA, it is known that in some cases, addition of BSA can restore qPCR efficiency from zero to something: (https://pubmed.ncbi.nlm.nih.gov/21219370/)

"BSA was shown to restore positive signals in five different RT-qPCR systems that were otherwise completely inhibited by produce rinsate extracts."

But in other cases, some literature just describes BSA addition being helpful only when used with organic solvents like DMSO.

If we need to repeat the same reaction, we would need to set up something similar enough.

One way to do that would be to modify the recipe of ours as below:

Translating this recipe to NEB Luna 4X mix + UDG:

For EPC, Added 3.5 µL of EPC and then added 5.25 µL of water to top up to 8.75 µL of volume.

So this would allow me to test for 14 different samples (& NTC and EPC) in a multiplexed fashion.

Used the thermocycling program for M3019

Note: Since this was using the NEB Luna mix, ROX normalization was used.

N gene primer and probe (FAM) gave signal at Cq = 35. Not sure if this is an issue unique to NEB mix

Perfect EPC behavior

Seeing more variability in sapmles from Exeter generally and doesn't appear to show differences

RPP30 (VIC) unreliable across all RNA samples

Beta-actin (Cy5) also worked well this time and in fact worked better because no signal coming out from NTC.

E gene primer and probe (Texas Red) generally work ok across SEPA samples but worked less well on Exeter RNA samples.

Rewrote observations for clearer explanations:

Among all primers and probes, N gene 1 was probably the most reliable in checking for SARS-CoV-2 RNA in wastewater RNA samples.

Primers and probes targeting E gene had difficulty giving consistent signals from wastewater RNA samples

Could not amplify anything from S gene using any of the primer probe sets, even if they generally worked ok with EPC.

The point is: these samples were from Jan to Feb 2021, i.e. mostly the alpha variants, that's fine for 69-70_WT to not work but it didn't make sense for the other two K417_WT (WT for beta) and E484_WT (WT for beta and gamma) to fail.

Primer and probes targeting ACTB gave strong signal in EPC. Could be contamination. This is where EPC started to behave inconsistently and deviated from previous results.

Also, primers and probes targeting ACTB did not consistently report human ACTB RNA in wastewater samples.

Primers and probes targeting RPP30 gave no amplifications in any of the samples, thus RPP30 tracking might not be a good way to track human RNA in wastewater samples.

Inspection of the raw data curves (ROX uncorrected amplification curves) suggested that the above observations (and deviation from the last result) were not due to introduction of ROX.

Rewrote observations for clearer explanations and highlighting key points:

Switching to NEB RT-qPCR mix appeared to have improved consistency between technical replicates

In this run, EPC behaved as expected

Under this experimental condition, primers and probes targeting RPP30 appeared to be performing worse than primers and probes targeting ACTB in reporting human RNA material

(If we assume cDNA for ACTB and RPP30 would be present at more or less similar levels)

Also under this experimental condition, primers and probes targeting E gene appear to perform better than those for N gene,

because there was no signal detectable from NTC, therefore detectable and consistent amplifications could be classified as SARS-CoV-2 positive

This experiment might suggest that for consistency (to generate data that agree with SEPA), and for reasons still unknown to us, it might be beneficial to stick with NEB RT-qPCR mixes.

It may be worthwhile to retest primers and probes for S gene using NEB mixes. However, primers and probes for S gene performed relatively poorly before and I do not expect this to change drastically with NEB mixes.

It might be best to stick with NEB RT-qPCR mixes to maximize consistent behavior and data agreement with SEPA.

However, we have not repeated enough trials to reach a solid conclusion.

PCR0007 was completed and closed.