Effects of Different Lysis Buffers of Nucleic Acid Purification Kit on the Stability of Influenza Virus RNA

Abstract

aims: Under suboptimal storage and transport conditions, influenza virus (flu-v) RNA is prone to degradation and lysis buffers from RNA extraction kits have a potential to stabilize RNA. The aim of this study was to investigate the effects of different lysis buffers on the stability of flu-v RNA. Materials & methods: Aliquots of flu-v suspension were processed in parallel with two lysis buffers, and then underwent cyclic freeze-thaw or prolonged storage at 4, 22 and -20°C. The viral RNA was analyzed by using real-time and conventional RT-PCR amplifying, respectively, partial and full-length sequences of the flu-v matrix gene. Results: The viral RNA remained intact in samples treated with either of the two lysis buffers for at least 7 days at 4°C, 90 days at -20°C or following seven freeze-thaw cycles, but buffer A was superior to buffer B in protecting RNA from degradation at 4°C and 22°C, or following a further increase of freeze-thaw cycles. conclusion: Lysis buffer preservatives provide viral RNA stabilization, whereas different lysis buffers vary in their ability to stabilize viral RNA, and thus their performance characteristics should be evaluated prior to their application in clinical practice. KeywordsNucleic acid (NA)-based techniques are now widely used in influenza surveillance and diagnosis [1,2]. However, suboptimal storage and transport conditions of influenza virus (flu-v) samples will most likely result in viral RNA degradation [3][4][5], and in some settings it is difficult to ensure certain sample conditions required by WHO, such as ultra-low-temperature freezing, especially for resource-limited areas; consequently, the efficacy of the NA-based assays will be compromised. Thus, inexpensive, simple and efficient sample stabilization methods need to be developed for facilitating flu surveillance and diagnosis.Earlier studies have shown that lysis buffers of NA extraction kits have the potential to be used as RNA stabilization agents at adverse temperatures and over an extended period of time [6,7], and have potential to inactivate viruses completely [8], which would reduce the biohazard risk from sample leakage during transit and simplify transportation requirements while preserving the integrity of viral RNA before testing. Nevertheless, these studies did not give a comprehensive picture of the abilities of lysis buffers in protecting viral RNA from degradation and there are still some questions that remain unanswered. First, could intact viral RNA be recovered from the samples preserved in lysis buffer at adverse temperatures and over an extended period of time, considering that a real-time RT-PCR (rRT-PCR) targeting a shorter fragment was only employed to evaluate the presence, quantity and/or quality of viral RNA in these studies [6,7]? Second, what is the consequence of virus samples preserved in lysis buffer and subjected to repeated freeze-thaw cycles, For reprint orders, please contact: reprints@futuremedicine.com

The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing

“…Interestingly, time-and temperature-dependant fragmentation was seen for these secondary products, in line with that seen for the target amplicons. The results of this study support the findings of Liu et al, 37 who were unable to amplify the entirety of a target influenza matrix gene after storage in an AVL buffer at 4 C for 15 to 45 days before extraction, although the viral RNA concentrations, as indicated by their RT-qPCR, were well within the detection limit of the assay.…”

“…This result is in contrast to previous findings that demonstrated storage in AVL protected viral RNA after seven freeze-thaw cycles. 37 However, as with other studies, sample integrity after freeze-thaw cycles was measured by RT-qPCR using a small target amplicon. This study saw no marked difference in the integrity of viral RNA from samples stored at À20 C or À80 C. This may be because of the length of time for which the samples were stored.…”

Stability of SARS-CoV-2 RNA in Viral Lysis Buffer Stored at Different Temperatures

“…Previous studies on other viruses established viral RNA integrity in viral lysis buffer kept at 20°C, 4°C and 25°C. 6,8 The study included 20 SARS-CoV-2 positive samples having both the lower Ct values (n = 10) and higher Ct values (n = 10) (Ct values ranging from 16 to 35). These known positive samples were lysed in parallel using viral lysis buffers and stored separately at 2 to 8°C and 22 to 28°C for 24 hours, 48 hours and 72 hours.…”

“…The use of viral lysis buffers to stabilize viral RNA has been investigated for viruses other than SARS-CoV-2. 5 6 7 For the first time, the present study investigated the potential of viral lysis buffer to stabilize SARS-CoV-2 virus RNA at varying temperatures and periods of time. Previous studies on other viruses established viral RNA integrity in viral lysis buffer kept at 20°C, 4°C and 25°C.…”

“…RNA extraction kits are provided with the viral lysis buffer and used commonly for their intended purpose of lysing cell, inactivating RNases and stabilizing RNA during the extraction process. [5][6][7] The potential of viral lysis buffer for SARS-CoV-2 RNA stabilization at varying temperatures and periods of time remains unknown. To find a practical solution, we evaluated the stability of SARS-CoV-2 RNA in viral lysis buffer of RNA extraction at J Lab Physicians:2020;12:268-270 two different temperature conditions by testing specimens on different time periods.…”