Application of Freeze-thaw Harvest for SARS-CoV-2

PCR EQA Panel Material

Nur Ika Hariastuti, Nike Susanti, Hana Apsari Pawestri and Kartika Dewi Puspa

Center for Research and Development of Biomedical and Basic Health Technology,

National Institute of Health Research and Development, Ministry of Health,

Jl. Percetakan Negara 23, Jakarta, Indonesia

Keywords: Freeze-thaw, SARS-CoV-2, Quality Assurance Panel.

Abstract: BACKGROUND: Currently there are more than 700 testing laboratories for COVID-19 in Indonesia. To

ensure that the laboratory has a good performance, a proficiency test panel for external quality assurance

program was conducted. The production of panels derived from virus isolate from cell culture, generally does

not contain ribonuclease protein (RNP) as in clinical samples. OBJECTIVE: To generate a panel that

resembles clinical samples, we conducted experiments to produce panels containing RNP by freeze-thaw

protocol. METHODS: SARS-CoV-2 virus cultures were performed in the NIHRD BSL-3 laboratory facility.

Harvesting is carried out on the 3rd, 6th, and 9th days with: no freeze-thaw process, 1 freeze-thaw process, 2

freeze-thaw processes, and 3 freeze-thaw processes. RESULTS: On the 3rd day of observation, the Ct isolates

had reached an average of 11.53 and did not increase with the increase in incubation time. Meanwhile, the

viral Ct became smaller in the presence of freeze-thaw treatment. RNP began to be detected on day 3 with an

average Ct of 35.40 and improving with the addition of days and the number of freeze-thaw treatments.

CONCLUSION: Freeze-thaw treatment can be used to improve the value of Ct however, the detected Ct RNP

value was still higher than the viral Ct.

1 INTRODUCTION

Severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2) was first discovered in 2019 in

Wuhan and caused an outbreak of pneumonia (Zhu,

Zhang, et al. 2020, Chan et al. 2020, WHO 2020b).

This virus spreads and causes a pandemic. SARS-

CoV-2 was first detected in Indonesia in March 2020

and at that time the laboratory designated as a testing

laboratory was only the Virology laboratory in the

Center for Research and Development for Biomedical

and Basic Health Technology (CRDBBHT), based on

Health Ministry Decree No. 658 concerning referral

laboratory networks for emerging and re-emerging

diseases (Health 2009).

Indonesia has a fairly high population density and

is spread across 5 major islands with various

transportation complexities. This of course affects the

speed of sending specimens to the Virology

laboratory CRDBBHT, which may cause delays in

diagnosis. For this reason, the government has

appointed government and private laboratories to

carry out laboratory tests of SARS-CoV-2 detection

in Indonesia using the real-time RT-PCR method.

Additionally, there are many reagents on the market

and have not been fully validated (WHO 2020a).

Apart from that, only a small proportion of the

laboratories that are designated as examination

laboratories have the ability to carry out examinations

using the PCR method. To ensure the accuracy of the

final results issued by the laboratory, quality control

is needed so that the results issued are accurate and

reliable. One of the elements needed in quality control

is external quality assurance (EQA) ((ASLM) 2020).

Materials used for testing the quality of COVID-

19 laboratory may come from clinical samples or

isolates obtained from viral cultures. SARS-CoV-2

has been successfully cultured and propagated using

cell culture, using both primary human airway

epithelial cells (AEC) and Vero African green

monkey kidney epithelial E6 cells (Barrow et al.

2021). Viral growth can be seen with the emergence

of damage to cells known as the cytopathic effect

(CPE). Most of the viruses leave the cell and are

found in the medium, but some viruses bind to the

cells. To excrete cell-bound viruses, a combination of

250

Hariastuti, N., Susanti, N., Pawestri, H. and Puspa, K.

Application of Freeze-thaw Harvest for SARS-CoV-2 PCR EQA Panel Material.

DOI: 10.5220/0010748800003113

In Proceedings of the 1st International Conference on Emerging Issues in Technology, Engineering and Science (ICE-TES 2021), pages 250-253

ISBN: 978-989-758-601-9

freeze-thaw, sonication, or a combination of both is

usually used (Hierholzer, Killington, and Stokes

1996, Laposova, Oveckova, and Tomaskova 2017).

Another way to get a high enough viral titer is by

performing passages (Mengesha et al. 2014).

One of the internal controls used to validate the

results of the PCR examination is the appearance of

an RNP curve which indicates the presence of the rnp

gene from human epithelial cells in the specimen

being examined. Rnp is a protein found in the nucleus

and cytoplasm. Some of the commercial panels used

for COVID-19 EQA do not contain RNP, so some

laboratories have concluded that the panels examined

were invalid. The invalid status referred to here is an

invalid sampling process, not the results of the

examination.

In the implementation of the proficiency test

organized by the Ministry of Health through the

National Institute of Health Research and

Development (NIHRD) in collaboration with WHO,

many laboratory participants encountered difficulties

in determining the test results. This is because the

RNP Ct did not appear from the panel test and caused

many laboratories did not to obtain a perfect result in

the test. To improve the performance of the COVID-

19 network laboratory in Indonesia, we are trying to

produce panels containing the rnp gene so that they

resemble clinical samples.

2 METHODS AND MATERIALS

In this study, we inoculated 3 SARS-CoV-2 isolates,

which are preserved biological materials belonging to

the national reference laboratory for COVID-19

testing under the ethical approval number

LB.02.01/2/KE.432/2020 from the NIHRD ethical

committee. Isolation was carried out with differences

in incubation time and the amount of freeze-thawed

process to see the differences of viral Ct and RNP.

2.1 Cell Line Preparation

Vero E6 cells used for virus isolation and propagation

were maintained using Dulbecco's modified Eagle

medium (DMEM) with the addition of 10% fetal

bovine serum (FBS), l-glutamine 2 mM, gentamicin,

100 units of Penicillin, and 100 µg / mL of

Streptomycin. Cells were grown in a BSL-2

laboratory using TC Flask 25cm

and incubated in an

incubator at 37

C and 5% CO2 for 1-2 days until it

reaches 90% confluent.

2.2 SARS‑CoV-2 Virus Inoculation

The SARS-CoV-2 cultures were conducted in the

BSL-3 facility. When the cells were 90% monolayer,

the medium in TC Flask 25cm

was discarded and

washed with 3 mL of PBS 2 times and then added 200

µL of SARS-CoV-2 isolate. The cultured specimens

were then incubated in an incubator at 37

C and 5%

for 60 minutes. Then added with the viral culture

medium used containing DMEM with 2% fetal

bovine serum (FBS), 2 mM l-glutamine, gentamicin,

100 units of Penicillin, and 100 ug / mL of

Streptomycin were added to the TC flask. The flasks

were incubated in an incubator at 37

C and 5% CO2

for 0, 3, 6, and 9 days.

2.3 RNP Detection

A total of 500 µL of isolates were taken from each

flask and put in a micro-centrifuge tube as samples

without freeze-thaw treatment. The TC flask was put

into the -70oC deep freezer until frozen (1st freeze),

then removed from the freezer and allowed to run

until it is liquid (1st thaw). Furthermore, as many as

500 µl isolates were taken and transferred into a

microcentrifuge tube as a sample for one freeze-thaw

treatment. The flasks were placed in the deep freezer

again until the culture freeze for the 2nd time and

removed from the freezer to get the 2nd thaw. We

repeat the steps to obtain 3 freeze-thaw cycles.

Isolates were extracted manually by the spin

column method using the QIAamp Viral RNA Mini

Kit (Qiagen) and the rnp gene was carried out using a

Biorad CFX96 with a set of primer forward (AGA

TTT GGA CCT GCG AGC G) reverse (GAG CGG

CTG TCT CCA CAA GT) and probe (TTC TGA

CCT GAA GGC TCT GCG CG) in the realtime RT-

PCR method using Real-Q 2019-nCoV (Biosewoom)

according to the manufacturer's protocol (WHO

2009).

The data obtained were then analyzed

quantitatively descriptively to determine the freeze-

thaw process against the Ct of viral and rnp genes.

3 RESULTS AND DISCUSSION

Based on the observation, it is known that the three

SARS-CoV-2 virus isolates on the 3rd-day culture

incubation have shown CPE or cell morphological

damage when compared to the control. Observations

using scanning electron microscopy (SEM)

conducted by Zhu et al., showed the formation of

plaque-like CPE that continues to expand in cells

Application of Freeze-thaw Harvest for SARS-CoV-2 PCR EQA Panel Material

251

infected with the SARS-CoV-2 virus as the

incubation time increases (Zhu, Wang, et al. 2020).

The results of virus examination using the real-

time RT-PCR method showed a significant increase

in the number of viruses, from the initial average Ct

29.29 to 11.53. The Ct values did not differ

significantly in the addition of the 6- and 9-day

incubation periods, namely 12.68 and 12.11 (Figure

1). Therefore, it is known that incubation for 3 days

is optimal in producing the SARS-CoV-2 virus.

The freeze-thaw treatment which is intended to

break down cell tissue is known to increase the

number of viruses harvested. However, in this

experiment, the Ct value of the virus did not change

significantly (Figure 2). This may be due to the

majority of viral virions are already outside of the cell

since the first observation (3 x 24 hours).

Figure 1: The viral Ct in different incubation time.

Figure 2: The viral Ct in different number of freeze thaw

treatment.

Unlike the case with Ct virus, which was not

significantly different from the increase in incubation

time. In Figure 3 we can see that Ct for rnp has

increased with increasing incubation time. Initially,

even the rnp was not detected or the Ct was greater

than the cut-off value. However, on the last day of

observation, the average value was 22.66.

Figure 3: The RNP Ct detected in different incubation time.

In Figure 4, we can see that the Ct for rnp has

improved in the presence of the freeze-thaw

treatment. However, the best Ct can be obtained by 2

times freeze-thaw process, even though some studies

showed that 3-5 cycles of freeze-thaw would increase

the virus yield (Kong, Foster, and Foster 2008, Gupta

et al. 1996).

Figure 4: The rnp Ct detected in different incubation time.

The trend continues to improve for rnp detection

with the addition of days. However, for the freeze-

thaw treatment, it seems that the addition of freeze-

thaw cycles more than 2 times did not cause a

different impact up to 9 days of incubation.

The number of viruses produced was good enough

on day 3 (detected Ct: 11.53). However, the number

of rnp produced up to day 9 was still in the

intermediate level (detected CT: 22.66). Thus, in one

sample a balanced Ct condition has not been obtained.

The limitation of this study is that we did not observe

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

252

the effect of incubation time and freeze-thaw process

earlier than 3 days and later than 9 days of incubation.

Another study on the freeze-thaw aspect of RNA

detection by PCR method was carried out on the

dengue virus. The results showed that the freeze-thaw

process did not affect RNA detection (Anwar et al.

2009). Therefore, this method is promising for

continuous development to obtain optimal Ct. It is

necessary to carry out a further study with a longer

incubation time to determine the best conditions for

producing a balanced Ct number between viral genes

and rnp.

4 CONCLUSIONS

The process of harvesting the SARS-CoV-2 virus

culture by the freeze-thaw process is sufficient for an

incubation period of 3 days and can be used for

producing EQA panel material. However, the

detected Ct rnp value was still higher than the viral

Ct. Thus, it is necessary to carry out further

optimization in the process of harvesting the SARS-

CoV-2 virus as a panel material for proficiency

testing that resembles clinical specimens.

ACKNOWLEDGEMENTS

The authors would like to thank the Director of

CDRBBHT and all the team members of SARS-CoV-

2 virus testing at the National Reference Laboratory

for COVID-19.

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