SARS-CoV-2 UK, South African and Brazilian Variants in Karachi- Pakistan

The coronavirus disease 2019 (COVID-19) has caused and is still causing tremendous damage to the global economy and human health. Qualitative reverse transcription-PCR (RT-qPCR) is the golden standard for COVID-19 test. However, the SARS-CoV-2 variants may not only make vaccine less effective but also evade RT-qPCR test. Here we suggest an innovative primer design strategy for the RT-qPCR test of SARS-CoV-2. The principle is that the primers should be designed based on both the nucleic acid sequence and the structure of the protein encoded.

The three nucleotides closest to the 3′ end of the primer should be the codon which encodes the tryptophan in the structure core. Based on this principle, we designed a pair of primers targeting the nucleocapsid (N) gene. Since tryptophan is encoded by only one codon, any mutation that occurs at this position would change the amino acid residue, resulting in an unstable N protein.

This means that this kind of SARS-CoV-2 variant could not survive. In addition, both our data and previous reports all indicate that the mutations occurring at other places in the primers do not significantly affect the RT-qPCR result. Consequently, no SARS-CoV-2 variant can escape detection by the RT-qPCR kit containing the primers designed based on our strategy.

The aim of the present study is to apply the canine olfactory sensitivity to detect COVID-19-positive axillary sweat samples as a One Health approach in Latin America. One hundred volunteers with COVID-like symptoms were invited to participate, and both axillary sweat samples for dog detection and nasopharynx/oropharynx swabs for qPCR were collected.

Two dogs, previously trained, detected 97.4% of the samples positive for COVID-19, including a false-negative qPCR-test, and the positive predictive value was 100% and the negative predictive value was 98.2%. Therefore, we can conclude that canine olfactory sensitivity can detect a person infected with COVID-19 through axillary sweat successfully and could be used as an alternative to screen them without invasive testing.

Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology.

In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E).

Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2-3 PCR-cycle threshold (Ct) values less when compared to samples stored at -20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%).

Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA transmission route was thoroughly investigated in the hospital wastewater, sewage collection network, and wastewater treatment plants. Samples were taken on four occasions from December 2020 to April 2021. The performance of two different wastewater treatment processes of sequencing batch reactor (SBR) and conventional activated sludge (CAS) was studied for virus destruction. For this purpose, liquid phase, solid phase and bioaerosol samples were taken from different units of the investigated wastewater treatment plants (WWTPs).

The results revealed that all untreated hospital wastewater samples were positive for SARS-CoV-2 RNA. The virus detection frequency increased when the number of hospitalized cases increased. Detection of viral RNA in the wastewater collection system exhibited higher load of virus in the generated wastewater in areas with poor socioeconomic conditions. Virus detection in the emitted bioaerosols in WWTPs showed that bioaerosols released from CAS with surface aeration contains SARS-CoV-2 RNA posing a potential threat to the working staff of the WWTPs. However, no viral RNA was detected in the bioaerosols of the SBR with diffused aeration system.

Green qPCR SuperMix UDG
20-abx098032	Abbexa	EUR 1362.00 EUR 543.60 EUR 794.40	15 ml 1 ml 5 ml

Top Green qPCR SuperMix
20-abx098033	Abbexa	EUR 994.80 EUR 260.40 EUR 1362.00 EUR 493.20	15 ml 1 ml 25 ml 5 ml

Tip Green qPCR SuperMix
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qPCR Control Template Kit
G929	ABM	100 rxns	EUR 177.6

Lentivirus qPCR Quantification Kit
K1471-100	Biovision	100 Rxns	EUR 357.6

Retrovirus qPCR Quantification Kit
K1472-100	Biovision	100 Rxns	EUR 381.6

Eva QPCR SuperMix Kit
K5052200	Biochain	200 reactions	EUR 253.2
Description: Premade ready to use kits will always come in handy. Get your experiment done right form the first try by using a validated kit with perfectly balanced reagents proportions and compatibility and by following a clear protocol.

Eva QPCR SuperMix Kit
K5052400	Biochain	400 reactions	EUR 384
Description: Premade ready to use kits will always come in handy. Get your experiment done right form the first try by using a validated kit with perfectly balanced reagents proportions and compatibility and by following a clear protocol.

Pro QPCR SuperMix Kit
K5053200	Biochain	200 reactions	EUR 225.6
Description: Premade ready to use kits will always come in handy. Get your experiment done right form the first try by using a validated kit with perfectly balanced reagents proportions and compatibility and by following a clear protocol.

Pro QPCR SuperMix Kit
K5053400	Biochain	400 reactions	EUR 358.8
Description: Premade ready to use kits will always come in handy. Get your experiment done right form the first try by using a validated kit with perfectly balanced reagents proportions and compatibility and by following a clear protocol.

Neural Lineage qPCR Profiler
RA500A-1	SBI	10 reactions	EUR 1198.8

Cancer MicroRNA qPCR Array
RA610A-1	SBI	10 profiles	EUR 1406.4

Regulatory RNA qPCR Profiler
RA950A-1	SBI	20 rxn	EUR 1056

Entrans qPCR Probe Kit
RK21209	Abclonal	40 RXN	Ask for price

Entrans qPCR Probe Set
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QPCR Kit DNA Staphylococ
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QPCR Kit DNA Bifidobacte
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QPCR Kit DNA Clostridium
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QPCR Kit DNA Lactobacill
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QPCR Kit DNA Anacardium
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QPCR Kit DNA Bertholleti
MOL6498	Scientific Laboratory Supplies	EACH	EUR 876.66

Investigation of SARS-CoV-2 RNA in WWTPs showed high affinity of the virus to be accumulated in biosolids rather than transporting via liquid phase. Following the fate of virus in sludge revealed that it is completely destructed in anaerobic sludge treatment process. Therefore, based on the results of the present study, it can be concluded that receiving water resources could not be contaminated with virus, if the wastewater treatment processes work properly.