Room-temperature-storable PCR mixes for SARS-CoV-2 detection

Xu, Jiasu; Wang, Jin; Zhong, Zecheng; Su, Xiaosong; Yang, Kunyu; Chen, Zhongfu; Zhang, Dongxu; Li, Tingdong; Wang, Yingbin; Zhang, Shiyin; Ge, Shengxiang; Zhang, Jun; Xia, Ningshao

doi:10.1016/j.clinbiochem.2020.06.013

Cited by 27 publications

(22 citation statements)

References 23 publications

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“…[28,47] Moreover, most products are provided as a lyophilized premix rather than liquid and separated components. [48][49][50][51] Compared with liquid reagents, lyophilized premix products have two advantages. First, lyophilized reagents can be preserved at room temperature, which decreases the requirement for cold chain transportation and storage.…”

Section: Poctmentioning

confidence: 99%

Nucleic Acid Testing for Coronavirus Disease 2019: Demand, Research Progression, and Perspective

Zhang

Wang

et al. 2020

Critical Reviews in Analytical Chemistry

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The current coronavirus disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a public health emergency of international concern. There has been a surge in demand for COVID-19 diagnostic reagents, as timely detection of virus carriers is one of the most important components of disease prevention and control. Nucleic acid testing (NAT), with high sensitivity and specificity, is considered the "gold standard" for the diagnosis of COVID-19. Therefore, more than 700 research units and companies have been devoted to developing NAT reagents. To date, nearly 600 research units and companies have claimed to have completed the development of NAT reagents. The use of these products has a positive effect on disease prevention and control; however, exaggerated claims and inadequate understanding of the products have led to improper access to reagents and equipment in clinics. This has resulted in chaos in the clinical diagnosis of COVID-19. Herein, we have overviewed the COVID-19 NAT products, including their principles, corresponding advantages and disadvantages, relevant circumstances for application, and respective roles in epidemic containment. Our comments may provide some references for assay developers and aid clinical staff in choosing the appropriate class of test from the different tests available.

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Section: Poctmentioning

confidence: 99%

Nucleic Acid Testing for Coronavirus Disease 2019: Demand, Research Progression, and Perspective

Zhang

Wang

et al. 2020

Critical Reviews in Analytical Chemistry

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“…Several publications on freeze-dried PCR mixes have been reported during the past 20 years ( Table S1 ) [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] ]. In previous studies, electrophoresis has frequently been used to evaluate the activities of freeze-dried PCR reagents [ 9 , 10 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

“…1 Ciii). However, most freeze-dried PCR reagents detected by qPCR have demonstrated poor stability during long-term storage at RT [ 11 , 14 , 16 ], especially those designed for RNA targets detection [ 15 , 21 ], which contain the thermally unstable reverse transcriptase [ 22 , 23 ]. And what's worse, currently, most PCR reagents are freeze-dried in PCR tube strips and cannot be transferred (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Several relevant publications have attempted to use elevated temperatures in accelerated stability testing to shorten the evaluation period for freeze-dried PCR reagents (Fig. 1Dv and Table S1 ) [ 10 , 14 , 21 ]. However, the temperatures they selected were incomplete, and the observation periods they reported were relatively narrow due to the poor freeze-drying methods used for their PCR reagents.…”

Section: Introductionmentioning

confidence: 99%

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Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics

Wang

et al. 2021

Talanta

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As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18–25 °C) for 1–2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q 10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.

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“…Finally, most SARS-CoV-2 testing protocols in clinical use or in pre-clinical development rely on commercial one-step RT-qPCR master mixes [5,13,18,19,[26][27][28][29][30][31][32]. However, the high cost of commercial master mixes could be prohibitive for widespread testing in resource-limited settings.…”

Section: Introductionmentioning

confidence: 99%

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Graham

Dugast-Darzacq

Dailey

et al. 2020

Preprint

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Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.

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Room-temperature-storable PCR mixes for SARS-CoV-2 detection

Cited by 27 publications

References 23 publications

Nucleic Acid Testing for Coronavirus Disease 2019: Demand, Research Progression, and Perspective

Nucleic Acid Testing for Coronavirus Disease 2019: Demand, Research Progression, and Perspective

Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

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