A Cold Chain-Independent Specimen Collection and Transport Medium Improves Diagnostic Sensitivity and Minimizes Biosafety Challenges of COVID-19 Molecular Diagnosis

Saini, Vikram; Kalra, Priya; Sharma, Manish; Rai, Chhavi; Saini, Vikas; Gautam, Kamini; Bhattacharya, Sankar; Mani, Shailendra; Saini, Kanchan; Kumar, Sunil

doi:10.1128/spectrum.01108-21

Cited by 8 publications

(9 citation statements)

References 26 publications

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“…The RNA was extracted from PBMCs using TRIzol reagent as per well-established protocols (1,3,23). RNA concentration (ng/mL) and purity (A260/A280) were determined spectroscopically (Cyatation Biotek, USA).…”

Section: Methodsmentioning

confidence: 99%

“…However, most of these genes do not consistently manifest stable expression under various experimental conditions (19)(20)(21). This could be especially critical in the context of SARS-CoV-2 infection since it manifests a wide spectrum of disease severity ranging from asymptomatic, mild, moderate and severe; each with a distinct clinical features/parameter (22)(23)(24). Moreover, during SARS-CoV-2 infection or following recovery, there can be acute co-infections especially that of fungal (mucormycosis) which may further impact the stability and expression of the appropriate reference gene.…”

Section: Introductionmentioning

confidence: 99%

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Selection of appropriate reference genes for normalization of qRT-PCR based gene expression analysis in SARS-CoV-2, and Covid-associated Mucormycosis infection

Kumar

Ahmad

Kushwaha

et al. 2022

Preprint

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Selection of reference genes in quantitative PCR is critical to determine accurate and reliable mRNA expression. Despite this knowledge, not a single study has investigated the expression stability of housekeeping genes to determine their suitability to act as reference gene in SARS-CoV-2 or Covid19 associated mucormycosis (CAM) infections. Herein, we address these gaps by investigating the expression stability of nine most commonly used housekeeping genes including TBP, CypA, B2M, 18S, PGC-1α, GUSB, HPRT-1, β-ACTIN and GAPDH in the patients of varying severity (asymptomatic, mild, moderate and severe). We observed significant differences in the expression of candidate genes across the disease spectrum. Next, using various statistical algorithms (delta Ct, Normfinder, Bestkeeper, RefFinder and GeNorm), we observed that CypA demonstrated the most consistent expression across the patients of varying severity and emerged as the most suitable gene in Covid19, and CAM infections. Incidentally, the most commonly used reference gene GAPDH showed maximum variations and found to be the least suitable. Lastly, comparative evaluation of expression of NRF2 is performed following normalization with GAPDH and CypA to highlight the relevance of an appropriate reference gene. Our results reinforce the idea of a selection of housekeeping genes only after validation especially during infections.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection of appropriate reference genes for normalization of qRT-PCR based gene expression analysis in SARS-CoV-2, and Covid-associated Mucormycosis infection

Kumar

Ahmad

Kushwaha

et al. 2022

Preprint

Self Cite

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“…The media was able to inactivate the virus even at 50% dilution as analysed by the absence of plaque formation. The samples transported in SSTM showed lower Ct value (mean Ct difference >3.50) and 70% higher detection of positive samples compared to the VTM used as reference [ 24 ].…”

Section: Viruses and Transport Mediummentioning

confidence: 99%

From cold chain to ambient temperature: transport of viral specimens- a review

Dsa,

Kadni,

2023

Annals of Medicine

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The diagnosis of an aetiology is dependent on the collection, transport, and storage of the infectious sample. The transport of the sample plays a crucial role in the chain of diagnosis. It is important to maintain the biological integrity of the pathogen during the transport of the sample to achieve an accurate diagnosis. This is important, particularly for labile organisms like viruses that are inactivated easily compared to other microorganisms. Many transport media have been utilised to ensure the integrity of the virus during transport. While most of the transport media are focused on preserving the infectious properties of the virus, progress has been made to develop virus transport media to inactivate the virus and obtain the stability of the viral nucleic acid, enabling better molecular diagnosis of the virus aetiologies. This review summarises the various media used for the transport of virus samples and focuses on the need to develop virus transport media that inactivates the virus and preserves the viral nucleic acid.

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“…Active surveillance testing requires the shipment of suspected samples of highly contagious diseases to BSL3+ high containment facilities for PCR confirmatory testing to meet select agent requirements ( 12 , 13 ). The shipment of potentially infectious substances is costly and time-sensitive and it requires specialized shipping containers and a cold chain to avoid the deterioration of samples for PCR testing ( 14 ). An inactivation method that protects the nucleic acid content essential for PCR testing while removing the threat of transmission and minimizing the temperature requirement is urgently needed to minimize shipping costs and derestrict confirmatory testing to lower biosafety-level facilities (BSL2).…”

Section: Introductionmentioning

confidence: 99%

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics

Welch,

Shrestha,

Hutchings

et al. 2024

Front. Vet. Sci.

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There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

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A Cold Chain-Independent Specimen Collection and Transport Medium Improves Diagnostic Sensitivity and Minimizes Biosafety Challenges of COVID-19 Molecular Diagnosis

Cited by 8 publications

References 26 publications

Selection of appropriate reference genes for normalization of qRT-PCR based gene expression analysis in SARS-CoV-2, and Covid-associated Mucormycosis infection

Selection of appropriate reference genes for normalization of qRT-PCR based gene expression analysis in SARS-CoV-2, and Covid-associated Mucormycosis infection

From cold chain to ambient temperature: transport of viral specimens- a review

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics

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