URL: https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973 TITLE: Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics ERROR: None [Skip to main content](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#main-content) ![Image 1](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/us_flag.svg) An official website of the United States government Here's how you know Here's how you know ![Image 2](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/icon-dot-gov.svg) **Official websites use .gov** A **.gov** website belongs to an official government organization in the United States. ![Image 3](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/icon-https.svg) **Secure .gov websites use HTTPS** A **lock** ( ) or **https://** means you've safely connected to the .gov website. 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Learn more: [PMC Disclaimer](https://pmc.ncbi.nlm.nih.gov/about/disclaimer/) | [PMC Copyright Notice](https://pmc.ncbi.nlm.nih.gov/about/copyright/) ![Image 14: Talanta logo](https://cdn.ncbi.nlm.nih.gov/pmc/banners/logo-pheelsevier.png) Talanta . 2021 Aug 10;235:122797. doi: [10.1016/j.talanta.2021.122797](https://doi.org/10.1016/j.talanta.2021.122797) * [Search in PMC](https://pmc.ncbi.nlm.nih.gov/search/?term=%22Talanta%22[jour]) * [Search in PubMed](https://pubmed.ncbi.nlm.nih.gov/?term=%22Talanta%22[jour]) * [View in NLM Catalog](https://www.ncbi.nlm.nih.gov/nlmcatalog?term=%22Talanta%22[Title%20Abbreviation]) * [Add to search](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973?term=%22Talanta%22[jour]) # Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics [Jiasu Xu](https://pubmed.ncbi.nlm.nih.gov/?term=%22Xu%20J%22[Author]) ### Jiasu Xu a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China Find articles by [Jiasu Xu](https://pubmed.ncbi.nlm.nih.gov/?term=%22Xu%20J%22[Author]) a 1, [Jin Wang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Wang%20J%22[Author]) ### Jin Wang a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China b School of Life Sciences, Xiamen University, Xiamen, 361102, China Find articles by [Jin Wang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Wang%20J%22[Author]) a b 1, [Xiaosong Su](https://pubmed.ncbi.nlm.nih.gov/?term=%22Su%20X%22[Author]) ### Xiaosong Su c Xiang'an Hospital of Xiamen University, Xiamen, 361102, China Find articles by [Xiaosong Su](https://pubmed.ncbi.nlm.nih.gov/?term=%22Su%20X%22[Author]) c, [Guofu Qiu](https://pubmed.ncbi.nlm.nih.gov/?term=%22Qiu%20G%22[Author]) ### Guofu Qiu d Xiamen Innovax Biotech CO., LTD., Xiamen, 361022, China Find articles by [Guofu Qiu](https://pubmed.ncbi.nlm.nih.gov/?term=%22Qiu%20G%22[Author]) d, [Qiurong Zhong](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhong%20Q%22[Author]) ### Qiurong Zhong e Haicang Hospital of Xiamen, Xiamen, 361026, China Find articles by [Qiurong Zhong](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhong%20Q%22[Author]) e, [Tingdong Li](https://pubmed.ncbi.nlm.nih.gov/?term=%22Li%20T%22[Author]) ### Tingdong Li a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China Find articles by [Tingdong Li](https://pubmed.ncbi.nlm.nih.gov/?term=%22Li%20T%22[Author]) a, [Dongxu Zhang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhang%20D%22[Author]) ### Dongxu Zhang a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China Find articles by [Dongxu Zhang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhang%20D%22[Author]) a, [Shiyin Zhang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhang%20S%22[Author]) ### Shiyin Zhang a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China Find articles by [Shiyin Zhang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhang%20S%22[Author]) a∗, [Shuizhen He](https://pubmed.ncbi.nlm.nih.gov/?term=%22He%20S%22[Author]) ### Shuizhen He e Haicang Hospital of Xiamen, Xiamen, 361026, China Find articles by [Shuizhen He](https://pubmed.ncbi.nlm.nih.gov/?term=%22He%20S%22[Author]) e∗∗, [Shengxiang Ge](https://pubmed.ncbi.nlm.nih.gov/?term=%22Ge%20S%22[Author]) ### Shengxiang Ge a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China Find articles by [Shengxiang Ge](https://pubmed.ncbi.nlm.nih.gov/?term=%22Ge%20S%22[Author]) a, [Jun Zhang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhang%20J%22[Author]) ### Jun Zhang a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China Find articles by [Jun Zhang](https://pubmed.ncbi.nlm.nih.gov/?term=%22Zhang%20J%22[Author]) a, [Ningshao Xia](https://pubmed.ncbi.nlm.nih.gov/?term=%22Xia%20N%22[Author]) ### Ningshao Xia a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China b School of Life Sciences, Xiamen University, Xiamen, 361102, China Find articles by [Ningshao Xia](https://pubmed.ncbi.nlm.nih.gov/?term=%22Xia%20N%22[Author]) a b * Author information * Article notes * Copyright and License information a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China b School of Life Sciences, Xiamen University, Xiamen, 361102, China c Xiang'an Hospital of Xiamen University, Xiamen, 361102, China d Xiamen Innovax Biotech CO., LTD., Xiamen, 361022, China e Haicang Hospital of Xiamen, Xiamen, 361026, China Joint first authors. ✉ ∗Corresponding author. ✉ ∗∗Corresponding author. Received 2021 Jun 12; Revised 2021 Aug 4; Accepted 2021 Aug 7 © 2021 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. [PMC Copyright notice](https://pmc.ncbi.nlm.nih.gov/about/copyright/) PMCID: PMC8353973 PMID: [34517655](https://pubmed.ncbi.nlm.nih.gov/34517655/) ## [](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973)Abstract 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. ## [](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973)Abstract Image 1 **Keywords:**Microfluidic molecular diagnostics, Room-temperature-storable, Freeze-dried PCR mixes, Accelerated stability testing ## [](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973)Introduction 1 As the outbreak of coronavirus disease 2019 (COVID-19) [[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib1),[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib2)], on-site molecular diagnosis is becoming increasingly important [[3](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib3)]. Recent advances in point-of-care (POC) testing, especially microfluidic technology, make it possible to develop rapid, simple, cost-effective and portable molecular diagnostic tools on site [[4](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib4)]. However, traditional PCR reagents typically made for central laboratories are not applicable for microfluidic molecular diagnosis, unless a freeze-drying method is introduced. Firstly, traditional PCR reagents can't be stored at room temperature (RT) as the water molecules they contained drive many destabilization pathways [[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib5),[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib6)]. Freeze-drying allows for the preservation of activity in qPCR reagents over the long-term storage at RT because this process removes most of the water molecules [[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib7)]. Thus, microfluidic chip contained the freeze-dried reagents can be stored everywhere irrespective of local preservation conditions. Secondly, liquid-form PCR reagents are cumbersome and complicated to prepare, whereas freeze-dried reagents are convenient to use because they only require reconstitution in water. With reduced operating steps, the operational complexity, preparation time, pipetting errors [[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib8)] and errors associated with improper handling of wet reagents, requirements for the operating environment and personnel quality can all be reduced. Thirdly, instead of loading each component of PCR reagent to microfluidic chip separately, one can transfer all these components into the chip easily if they are freeze-dried as beads ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1)Aii). This can also simplify the design of microfluidic chip as only one chamber is needed to store all the components for PCR. Last but not least, microfluidic molecular diagnostics is typically used to manipulate small volume of liquids, including small volume of samples, which results in reduced detection sensitivity. Freeze-dried PCR mixes can make up for it if they are reconstituted with sample instead of water ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1) B).Fig. 1Schematic diagrams of the freeze-dried PCR mixes and related methods. (A) Freeze-dried PCR mixes. (i) The PCR reagents are freeze-dried in PCR tube strips and cannot be removed after freeze-drying. (ii) The PCR mixes are freeze-dried as a bead and can be transferred by tweezers after freeze-drying. (B) The detection sensitivity of microfluidic reagent is reduced by the smaller reaction volume, which can be made up by reconstituting the freeze-dried PCR mixes with more sample. (C) Residual activity evaluation of the freeze-dried PCR reagents. (iii) Activity of the reagents evaluated by qPCR. In this method, fluorescence was used to report the dynamic changes in nucleic acids during the PCR. (iv) Activity of the reagents evaluated by electrophoresis. In this method, only the final products were measured after PCR. (D) Accelerated stability testing can be used to shorten the evaluation period (v) and predict the shelf lives (vi) of freeze-dried PCR mixes. Higher temperatures are typically associated with shorter storage periods.Fig. 1 Several publications on freeze-dried PCR mixes have been reported during the past 20 years ([Table S1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#appsec1)) [[[9]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib9), [[10]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib10), [[11]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib11), [[12]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib12), [[13]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib13), [[14]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib14), [[15]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib15), [[16]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib16), [[17]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib17), [[18]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib18), [[19]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib19), [[20]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib20), [[21]](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib21)]. In previous studies, electrophoresis has frequently been used to evaluate the activities of freeze-dried PCR reagents [[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib9),[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib10),[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib12)]. However, the nucleic acids detected by electrophoresis represent the final products of the PCR, which are all the same when PCR reaches the plateau phase. Under this circumstance, the band intensities of PCR reagents with different levels of residual activity will appear the same in the electrophoresis assessment ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1)Civ). Quantitative real-time PCR (qPCR) is able to distinguish differences in reagent activity because it can monitor changes in nucleic acids throughout the PCR process. Using this method, the quantification cycle (Cq) values of degraded reagents would be larger than those with 100% activity ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1)Ciii). However, most freeze-dried PCR reagents detected by qPCR have demonstrated poor stability during long-term storage at RT [[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib11),[14](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib14),[16](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib16)], especially those designed for RNA targets detection [[15](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib15),[21](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib21)], which contain the thermally unstable reverse transcriptase [[22](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib22),[23](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib23)]. And what's worse, currently, most PCR reagents are freeze-dried in PCR tube strips and cannot be transferred ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1)Ai), which poses a greater challenge to current microfluidic diagnosis technology. Even if the freeze-drying method meets all of the requirements detailed above, the stability testing process over long periods under normal conditions for each production batch is time-consuming, labor-intensive, and not cost-effective. Generally speaking, biological reagents age faster when stored at higher temperatures. Several relevant publications have attempted to use elevated temperatures in accelerated stability testing to shorten the evaluation period for freeze-dried PCR reagents ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1)Dv and [Table S1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#appsec1)) [[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib10),[14](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib14),[21](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib21)]. 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. Also, even if an accelerated stability testing can be used to shorten the evaluation period, a real-time stability testing at RT remains necessary to establish the correlation between storage periods at higher temperatures and RT, which can still be time-consuming. To get rid of the real-time stability testing at RT, researches have attempted to translate the accelerated stability testing data into a predicted shelf life at RT using mathematical models ([Fig. 1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#fig1)Dvi and [Table S1](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#appsec1)) [[17](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib17),[18](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib18)]. However, in fact, no mathematical method was specially designed and developed to predict the shelf life of freeze-dried PCR reagents, and their accuracy has not been verified up to now. In this study, we have presented a freeze-drying method to generate transferable, easy-to-use and RT-storable PCR mixes that are suitable for microfluidic molecular diagnosis. Besides, we have introduced accelerated stability testing to shorten the evaluation period of these freeze-dried PCR mixes. In addition, mathematical models were also employed to predict the shelf life of the freeze-dried mixes, with further verification of their accuracy and potential influence factors. The results of this study would foster the development of molecular diagnoses in both central laboratories and resource-limited areas. ## [](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973)Materials and methods 2 ### Specimens 2.1 Enterovirus 71 (EV71), coxsackievirus A16 (CA16), human immunodeficiency virus (HIV), cytomegalovirus (CMV), hepatitis B virus (HBV), _Escherichia coli_ BL21 (_E._ _coli_), and the human hepatoma cells (HuH-7) were supplied by the National Institute of Diagnostics and Vaccine Development in Infectious Diseases (Xiamen, China). Before use, viruses were inactivated using appropriate methods for each virus. ### Nucleic acid extraction 2.2 Nucleic acids were extracted using Viral DNA/RNA Purification Kit, Bacteria DNA Purification Kit, or Tissue/Cell DNA Purification Kit with the DOF-9648 purification system (GenMagBio, China), according to the manufacturer's protocol. The extracted nucleic acids were stored in a 1.5-mL sample tube and maintained at −80°C before PCR. ### PCR assay 2.3 The 40-μL reactions and thermal cycling were the same as the ones described in our previous article [[21](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#bib21)], except for the sequence specific primers and probes ([Table S2](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973#appsec1)). ### Freezing step 2.4 [... middle omitted — see footer ...] * 23.Arezi B., Hogrefe H.. 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Rhythm._. 2008. 23(1):95-98. doi: 10.1177/0748730407311855 [[DOI](https://doi.org/10.1177/0748730407311855)] [[PMC free article](https://pmc.ncbi.nlm.nih.gov/articles/PMC2365757/)] [[PubMed](https://pubmed.ncbi.nlm.nih.gov/18258762/)] [[Google Scholar](https://scholar.google.com/scholar_lookup?journal=J.%20Biol.%20Rhythm.&title=Mammalian%20peripheral%20circadian%20oscillators%20are%20temperature%20compensated%20%5BJ%5D&author=Reyes%20B.A.&author=Pendergast%20J.S.&author=Yamazaki%20S.&volume=23&issue=1&publication_year=2008&pages=95-98&pmid=18258762&doi=10.1177/0748730407311855&)] # [](https://pmc.ncbi.nlm.nih.gov/articles/PMC8353973) Associated Data _This section collects any data citations, data availability statements, or supplementary materials included in this article._ ## Supplementary Materials Multimedia component 1 [mmc1.docx](https://pmc.ncbi.nlm.nih.gov/articles/instance/8353973/bin/mmc1.docx) * * * Articles from Talanta are provided here courtesy of ![Image 15: Close](https://cdn.ncbi.nlm.nih.gov/pmc/pd-medc-pmc-cloudpmc-viewer/production/a2b04810/var/data/static/img/usa-icons/close.svg) ## 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