Keywords
RNA - SARS-CoV-2 - viral lysis buffer
Introduction
The recent emergence of SARS-CoV-2 leads to an ongoing global health emergency.[1]
[2] During this pandemic, the incidence of cases is increasing rapidly. This resulted
in an increased need for diagnostic testing, using real-time PCR assays, by targeting
a set of different genes of the SARS-CoV-2 virus (ORF1ab, open reading frame 1a and b 226, N-gene [the nucleocapsid protein], E-gene [envelope protein], S-gene [spike protein] and RdRp gene [RNA-dependent RNA polymerase]).[3] Presently, testing laboratories are facing multiple challenges on different fronts,
including a sudden increase in sample load, which leads to delay of the specimen processing
and results in detection failure due to viral RNA degradation.[4] This leads to developing newer innovative strategies without compromising the quality
of testing outcome as well as biosafety. In this present situation, processing specimens
immediately and storing them under optimal conditions is not always possible. 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 two different temperature conditions by testing specimens on different time periods.
Materials and Methods
SARS-CoV-2 positive samples (n = 20) having Ct values ranging from 16 to 35 were selected for this evaluation. The
study was conducted primarily using viral lysis buffer of HiPurA Viral RNA purification
kit (Himedia, Mumbai, India); also, the following commercial RNA extraction kits
were used for further evaluation (i) Invitrogen Purelink RNA Mini Kit (Invitrogen,
USA), (ii) QIAamp Viral RNA Mini Kit (Qiagen, USA) and (iii) TRUPCR COVID-19 Viral
RNA Extraction kit (TRUPCR, India).
All the selected positive samples were processed parallelly in two sets of HiPurA
Viral RNA viral lysis buffer as per manufacturer’s instruction. Briefly, 140 μL aliquots
of the sample were added to 1.5 mL microcentrifuge tubes preloaded with aliquots of
the freshly prepared viral lysis buffer (560 μL), carrier RNA (5.6 μL) and MS2 phage
control (10 μL). On the other hand, 140 μL samples were kept in two parallel sets
of vials without addition of viral lysis buffer along with carrier RNA and MS2 phage
control. For the quality control aspect, MS2 phage control was used as an internal
process control of RNA extraction and the no lysis buffer control was used for the
comparison of samples added in lysis buffer.
All the prepared viral lysis buffer suspension and no lysis buffer vials were kept
at two different temperature conditions: refrigerator (2–8°C) and ambient temperature
(22–28°C) for 24 hours, 48 hours and 72 hours separately. Thereafter, RNA was extracted
from each of the experimental vials (samples suspensions [710 μL] + ethanol [560 μL])
using HiPurA Viral RNA purification kit (Himedia, Mumbai, India).
The integrity of extracted viral RNA was evaluated by multiplex real-time RT-PCR test
for the qualitative detection of RNA from SARS-CoV-2 using TaqPath RT-PCR COVID-19
kit (Applied Biosystems, CA, USA). This multiplexed assay contains three primer/probe
sets specific to different SARS-CoV-2 genomic regions (orf-1ab gene, gene for the S protein and gene for the N protein) and primers/probes for bacteriophage
MS2. The assay was performed on the QuantStudio 7 Pro Real-Time PCR System (Applied
Biosystems, CA, USA). Samples with discrepant results were retested in duplicate.
Furthermore, the same experiment was performed using the three other commercially
available RNA extraction kits to determine their performance characteristics in protecting
the viral RNA. The data were expressed as the mean ± standard deviation (SD) and analyzed
using MS Excel 2010.
Results
Mean and standard deviation of the Ct value of the three tested genes were calculated
for the analysis of the results. The variation of Ct values at 24 hours and 48 hours
was found to be in the accepted range of one SD interval of the initial testing Ct
values ([Table 1]). This indicates an unchanged quantity of SARS-CoV-2 viral RNA at 24 hours and 48
hours in both the temperatures. However, all the tested samples showed 1 or 2 Ct reduction
after incubation in lysis buffer for 24 hours in both the storage conditions. At 2
to 8°C, SARS-CoV-2 viral RNA was relatively more stable than room temperature storage.
Decline in the viral RNA quantity was observed in the samples stored up to 78 hours
in both the temperature conditions and 60% samples turned to be negative. No significant
difference in the stability of orf-1ab, N gene and S gene was noted. All three gene targets were found within the accepted range for up to
48 hours storage in both the temperatures. Samples placed in no lysis buffer did not
show positive results. Lysis buffers of other three commercially available RNA extraction
kits showed almost the similar results (data not shown).
Table 1
Stability of SARS-CoV-2 RNA on viral lysis buffer after a period of storage at different
temperatures
|
Storage temperature
|
2–8°C
|
22–28°C
|
|
Storage period
|
24 h
|
48 h
|
72 h
|
24 h
|
48 h
|
72 h
|
|
SARS-CoV-2 gene
|
Initial Ct value
(mean and SD)
|
Ct values
(mean)
|
|
orf-1ab gene
|
25.5 (± 9.50)
|
26.22
|
28.63
|
32.80
|
27.30
|
29.15
|
35.54
|
|
S gene
|
26.8 (± 6.85)
|
27.45
|
28.74
|
30.42
|
28.42
|
30.12
|
34.21
|
|
N gene
|
27 (± 5.10)
|
28.01
|
30.28
|
31.51
|
29.3
|
31.41
|
33.9
|
Discussion
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.[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. This study showed the integrity and reproducibility
of positivity in all the samples included in this study. The internal control MS2
phage was also demonstrated in all experimental setups.
The SARS-CoV-2 RNA remained intact in samples for at least 48 hours at 2 to 8°C and
22 to 28°C. Our results showed a significant correlation of initial testing Ct values
of orf-1ab, N gene and S gene of known SARS-CoV-2 sample compared with the experimental setup samples. Slight RNA
degradation was observed in samples stored at 22 to 28°C as compared with samples
stored in the refrigerator at 2 to 8°C.
The study included 10 samples with borderline Ct values, in which two samples had
Ct 35 for orf-1ab gene. All these borderline Ct values samples showed positive results when stored up to
48 hours. According to TaqPath multiplex RT-PCR COVID-19 kit protocol, any of the
two genes within Ct value of 36 and with the sigmoid amplification curve will be considered
as a positive. In this study, two samples with higher Ct value of 35 (for orf-1ab gene) showed 4 Ct reduction after incubation in lysis buffer for 48 hours. However, these
two samples were considered positive, as the other two genes (N gene and S gene) were in acceptable range and these samples turned negative after 72 hours incubation.
Higher variations in Ct values were also observed in samples stored for 72 hours’
time period at both temperatures. We did not further analyze samples for longer durations
(more than three days), as in this pandemic situation, delayed testing and reporting
seems worthless.
No positive results were observed from no lysis buffer controls and some were failed
in the RNA extraction procedure, as the internal MS2 phage control did not amplify
in PCR. Viral lysis buffers mainly contain chaotropic agents such as guanidine thiocyanate
(GITC) or other protein denaturants, which are sufficient to render viruses noninfectious
by their denaturing activity. Hence, samples without viral lysis buffer will not remain
suitable for diagnosis.
RNA extraction is a crucial step in the detection of SARS-COV-2 and the result outcome
depends upon the performance of the RNA extraction kit used. Different laboratories
are using different kits, and the kits are being changed very frequently, especially
during the current COVID-19 crisis. Hence, this study also evaluated other three commonly
used commercial RNA extraction kits for the ability to protect viral RNA. There was
no major difference in the Ct values, and all the kits showed almost similar results
by stabilizing RNA up to 48 hours.
The main purpose of this study was to evaluate the stability of SARS-CoV-2 viral
RNA and to evaluate the reproducibility and not to assess the quality and quantity
of the viral RNA.
Conclusion
In the recent emergence of SARS-CoV-2, testing laboratories are currently witnessing
increased sample load every day. Viral lysis buffer turns sample to be noninfectious
and provides stabilization of SARS-CoV-2 RNA for up to 48 hours even at room temperature.
This allows laboratories to maintain continuous workflow of sample processing and
testing without compromising timeliness and storage of samples.
