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Journal of Infectious Diseases & Therapy

ISSN: 2332-0877

Research Article

Open Access

Detecting and Isolating False Negatives of SARS-Cov-2 Primers and Probe
Sets among the Japanese Population: A Laboratory Testing Methodology
and Study
Wataru Tsutae1, Wirawit Chaochaisit1*, Hideyuki Aoshima1, Chiharu Ida1, Shino Miyakawa1, Hiroko Sekine1, Afzal Sheikh1,4, Iri Sato Baran1,
Toshiharu Furukawa1,2 and Akihiro Sekine1,3*
Genesis Institute of Genetic Research, Genesis Healthcare Corporation, Yebisu Garden Place, Shibuya-ku, Tokyo, Japan
Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan3
Department of Emergency and Critical Care Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, Japan
4
Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Dhaka, Bangladesh
12Abstract
Objective: The study was performed for a comparative analysis between primers from Japan’s and US’s disease control centers aswell as to investigate the virus sequence alignment with primers’ oligonucleotide to introduce primer sets of high detectability with reduce
false negative results.
Design or methods: 11,652 samples from Japanese population were tested for Novel Severe Acute Respiratory Syndrome Corona
Virus-2 (SARS-CoV-2) positive using recommended RT-PCR primer-probe sets from Japan National Institute of Infectious Disease (NIID)
and US Centers for Disease Control and Prevention (CDC). Primer-probe sensitivity was analyzed for higher detectability for SARSCoV-2 positive cases.
Results: Of the 102 positive samples, 17 samples (16.7% of total positives) showed inconsistent results when tested simultaneously
for the following primers: JPN-N2, JPN-N1, CDC-N1, and CDC-N2. Our results revealed that CDC recommended primer-probe sets
showed relatively higher detection sensitivity and accuracy. Further, virus sequence alignment analysis showed evidences for virus
mutation occurred at primer’s binding sites.
Conclusion: The inconsistency in the RT-PCR results for SARS-CoV-2 detection using JPN-N1, JPN-N2, CDC-N1, and CDC-N2
primer-probe sets could be attributed to differences in primer efficiency or/and virus mutation at primer-probe’ binding sites. The useof JPN-N2 combined with CDC-N2 and CDC-N1 primer-probe sets produce the most effective results as well as may reduce the false
negatives results in Japan and overseas.

Keywords: COVID-19; SARS-CoV-2; RT-PCR performance;
Genomic variants; Primers; Sensitivity

Introduction
The global COVID-19 pandemic has spread across various
continents in diverse methods and speed while opening up discussion
for technological and scientific questions pertaining methodology oftesting accuracy among diverse viral strains.
On the issue of testing sensitivity and accuracy, RT-PCR has
been the gold standard testing method for SARS-CoV-2 detection
compare to other rapid testing methods [1,2]. However, each country’s
infectious disease authority has established its own guidelines, primerprobe sets, and protocols, thereby; the global testing community lacks
a “Standardized Universal Primer(s)” (SUP) that is foolproof for the
COVID-19 patients among various populations today. As a result, RTPCR testing accuracy and results may vary depending on which primer
was used. Low primer efficiency or/and mutation at the primer-probe
binding sites as well as very low viral RNA in the sample may resulting
in false negative RT-PCR results.
There are two commonly known factors associated with inaccurate
testing results for COVID-19 using RT-PCR testing method. First is
the failure to retrieve sufficient amounts of viral RNA present in thesample is administered or the method of how a sample is collected. For
example, a nasopharyngeal swab may be unable to obtain sufficientamounts of RNA if it does not come in contact at a nasal position where
the presence of the virus is concentrated. In the case of saliva collection
kit, use during the first several days of viral contact may result ininsufficient amounts of RNA.
For both nasopharyngeal swab and saliva kit, low amounts of viralload occurring at later stage among discharged or recovering patientshas tendency to show extremely low RNA count. While many countries
have issued a standard 14-day quarantine, Genesis Healthcare, a

J Infect Dis Ther, an open access journal
ISSN: 2332-0877

licensed clinical laboratory in Tokyo, Japan, has confirmed throughtested samples where the recovering and discharged patient is still
testing positive after 14 day despite low RNA count. Though thisconfirmation does not necessarily imply that the patient is infectious
after 14 days of quarantine as observed in a study [3], while the test
result remains positive, it supports recent study which outlines thelong duration of the RNA-positive tail and calls for reconsideration ofcontainment strategy [4].
The second factor is the lack of a standardized international testing
method for COVID-19 using a set of common primer(s) among
different nations/populations or hereby referred to as SUP. Different
primers used by different country’s testing protocols prevent effective
tracking of pandemic due to differences in false negative results due toviral genetic variation which may have been introduced from different
regions.
Researchers have started to recognize this problem as more SARSCoV-2 genomic data are becoming available. In particular, a researchergroup evaluated five assay panels for possible loss of detection sensitivity*Corresponding author: Wirawit Chaochaisit, Genesis Institute of Genetic
Research, Genesis Healthcare Corporation, Yebisu Garden Place, Shibuya-ku,
Tokyo, Japan, E-mail: wirawit@genesis-healthcare.jp
Akihiro Sekine, Department of Emergency and Critical Care Medicine, Chiba
University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, Japan, E-mail: akihiro_
sekine@genesis-healthcare.jp
Received date: February 12, 2021; Accepted date: February 26, 2021; Publisheddate: March 05, 2021
Citation: Tsutae W, Chaochaisit W, Aoshima H, Ida C, Miyakawa S, et al. (2021)
Detecting and Isolating False Negatives of SARS-Cov-2 Primers and Probe Sets
among the Japanese Population: A Laboratory Testing Methodology and Study. J
Infect Dis Ther S1:004.
Copyright: © 2021 Tsutae W, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author andsource are credited.

Volume S1 • 1000004

Citation: Tsutae W, Chaochaisit W, Aoshima H, Ida C, Miyakawa S, et al. (2021) Detecting and Isolating False Negatives of SARS-Cov-2 Primers and Probe Sets among
the Japanese Population: A Laboratory Testing Methodology and Study. J Infect Dis Ther S1:004.

Page 2 of 6

due to genetic variability of the virus [5] which was consistent with thedata observed by Genesis Healthcare.
The current study of summarizing the RT-PCR result discrepancy
for SARS-CoV-2 testing among different primers recommended by
NIID and CDC will be of great interest to the scientific community
working on infectious diseases detection, diagnosis methods and healthcare.

JPN-N2 showed consistently higher primer sensitivity than
JPN-N1. As a result, it is sought that NIID later eliminated JPN-N1 to
only reflect JPN-N2 in calling positive samples.

Methods

Due to the continuing dialogue by the medical community toretest samples whose results were not considered to reflect patient’s
symptoms, testing JPN-N1 was continued and CDC-N1 and CDC-N2,
both of which are located in nucleocapsid region N of SARS-CoV-2,
were added to identify whether JPN-N2 primer was adequate to identify
positive cases for all samples.

Study Design

Sample Collection

Japan’s COVID-19 RT-PCR public health observation is unique inthat while Wuhan, China was locked down from January 23, 2020 till
April 8th, 2020; Japan’s border was still open to remainder of China
until March 9th, 2020 and to US and Europe until March 26, 2020.
This time gap in closing the international border leaves an inquiry that
COVID-19 could have possibly entered Japan at different times fromboth East and West.

All COVID-19 positive testing samples were obtained between
April 2020 and August 2020 and were either obtained from saliva or
nasopharyngeal test kits. The nasopharyngeal testing included two
swabs per person and were administered to maximize virus collection
to rule out miscalling due to low RNA count. Both nasopharyngeal
and saliva samples were immediately immersed in RNA preservation
solution after collection to inactivate the virus while maintaining
RNA stability during transport. Samples were delivered to the Genesis
Healthcare’s PCR testing facility within 24 hours at temperatures
between 20°C and 27°C. RNA extraction was immediately conductedfollowed by RT-PCR test. The entire RNA extraction to RT-PCR testingprocess was completed within 6 hours of receipt at the same laboratory
using the below testing method.

On March 11th and March 19th, NIID announced the testing
protocols for COVID-19 testing by outlining two primer-probe sets
JPN-N2 and JPN-N1 targeting the nucleocapsid region, N of SARSCoV-2 as shown in (Figures 1 and 2). The diversity sites were sourced
from [6].

Figure 1: Structural representation of SARS-CoV-2 and primer/probe sites.

Figure 2: A RT-PCR results for JPN-N2 and JPN-N1 primer-probe sets:
JPN-N2 shows relatively higher signal intensity compared to JPN-N1 and therising curve starts at 25 cycles for SARS-CoV-2 positive samples.

J Infect Dis Ther, an open access journal
ISSN: 2332-0877

Volume S1 • 1000004

Citation: Tsutae W, Chaochaisit W, Aoshima H, Ida C, Miyakawa S, et al. (2021) Detecting and Isolating False Negatives of SARS-Cov-2 Primers and Probe Sets among
the Japanese Population: A Laboratory Testing Methodology and Study. J Infect Dis Ther S1:004.

Page 3 of 6

COVID-19 RNA collection method, extraction and RT-PCR
testing method
Saliva samples were collected using saliva RNA sample collection
kit by Zeesan Biotech Co., Ltd. Nasopharyngeal swab samples werecollected using virus RNA sample collection kit by Zeesan Biotech Co.,
Ltd. To remove viscosity, 0.5 ml of 20% DTT was added to each saliva
sample followed by, vortex, incubation at 50° Celsius for 10 minutes,
and then centrifuged at 1,500 rpm for 10 minutes.
180 μl of supernatant of DTT-treated saliva samples and 180 μl of
nasopharyngeal swab suspension were used for RNA extraction. RNA
extraction was performed by MGISP-960 system (MGI Tech Co., Ltd).
RT-qPCR for specific amplification of the N gene or was performed
using Light Cycler 480 system Ⅱ (Roche Diagnostics K.K.). Final
reaction volume was 12.5 μl, including 2.0 μl of RNA template, 6.25 μlof One Step Prime Script III RT-qPCR Mix (TAKARA BIO INC.), andforward primer, reverse primer and probe (primer and probe sequence,
final concentration was shown in Table 1.
Primer/ probe set
JPN-N1JPN-N2

CDC-N1

CDC-N2

Primer name
JPN-N1-F
JPN-N1-R
JPN-N1-P
JPN-N2-F
JPN-N2-R
JPN-N2-P
CDC-N1-F
CDC-N1-R
CDC-N1-P
CDC-N2-F
CDC-N2-R

The cycling conditions consisted of RT at 50° Celsius for 5
minutes, initial denaturation at 95° Celsius for 10 sec, and 45 cycles ofdenaturation at 95° Celsius for 5 seconds and annealing/extension at
60° Celsius for 30 seconds.

Detectability of positive and negative case
The positive cased were judged by following rules: If the detection
signal arise at Ct (cycle threshold) values ≥ 20 but ≤ 35 were defined
as positive cases while at Ct values >35 were defined as weak positive
cases. Otherwise, all were defined as negative cases.

Results
All samples were tested simultaneously for JPN-N2, JPN-N1,
CDC-N2, and CDC-N1 primer/probe set (Note: As of May 2020, theJapanese testing protocol for COVID-19 is only JPN-N2 as announced
by the Japan’s NIID) (Table 2). Outlines that 85 samples (82 saliva
samples and 3 nasopharyngeal swab samples) of a total 102 samples
that were tested positive for JPN-N2 primer/probe set while 17 samples

Description
N_Sarbeco_F1
N_Sarbeco_R1
N_Sarbeco_P1
NIID_2019-nCOV_N_F2
NIID_2019-nCOV_N_R2
NIID_2019-nCOV_N_P2
CDC_2019-nCoV_N1-F
CDC_2019-nCoV_N1-R
CDC_2019-nCoV_N1-P
CDC_2019-nCoV_N2-F
CDC_2019-nCoV_N2-R

Oligonucleotide sequence (5’>3’)
CACATTGGCACCCGCAATCGAGGAACGAGAAGAGGCTTG
ACTTCCTCAAGGAACAACATTGCCA
AAATTTTGGGGACCAGGAACTGGCAGCTGTGTAGGTCAAC
ATGTCGCGCATTGGCATGGAGACCCCAAAATCAGCGAAATTCTGGTTACTGCCAGTTGAATCTG
ACCCCGCATTACGTTTGGTGGACCTTACAAACATTGGCCGCAAA
GCGCGACATTCCGAAGAA

Label
None
None
FAM, BHQ-1
None
None
FAM, BHQ-1
None
None
FAM, BHQ-1
None
None

Working conc.
0.48 mM
0.64 mM
0.16 mM
0.4 mM
0.56 mM
0.16 mM
0.5 mM
0.5 mM
0.2 mM
0.5 mM
0.5 mM

CDC_2019-nCoV_N2-P

ACAATTTGCCCCCAGCGCTTCAG

FAM, BHQ-1

0.2 mM

Table 1: List of primer-probe sets used in this study to check sensitivity and accuracy.

(15 saliva and 2 nasopharyngeal swab samples) were tested negative.

SalivaNasopharyngeal
swab
Total

JPN-N2
positive
82

JPN-N2
negative
15

3

2

5

85

17

102

Total
97

Table 2: Distribution of positive SARS-CoV-2 samples for JPN-N2.

However, Table 3 depicts that among the 17 samples which were
tested negative for JPN-N2, 12 samples were tested positive for CDC-N2
primer/probe set while negative for CDC-N1, 4 samples tested positive
for both CDC-N1 and CDC-N2. Furthermore, one sample was tested
positive for CDC-N1 while negative for CDC-N2 (Figures 3 a-3h).

SalivaNasopharyngeal swab
Total

CDC-N1positive
CDC-N2
negative
10
1

CDC-N1
negative
CDC-N2
positive
102
12

CDC-N1positive
CDC-N2
positive
4
0
4

Total
15
2
17

Table 3: Breakdown of JPN-N2 negative samples having positive response from
CDC-N1 and/or CDC-N2.

These 17 “test result irregularities”, which accounts for 16.7%
of total 102 positive sample pool would be declared negative if alaboratory only tested JPN-N2. This high rate of irregular occurrence
stemming from 1 primer/probe can be considered as one of the major
J Infect Dis Ther, an open access journal
ISSN: 2332-0877

causes leading to false negatives that are often reported in association
with COVID-19 testing accuracy. Furthermore, from the observation
above, a combination of multiple primers, in this case, a combinationof JPN-N2 and CDC-N2 primer shows the highest rate of accuracy
and sensitivity compared to JPN-N2 primer alone, followed by a triple
combination of JPN-N-2, CDC-N2 and CDC-N1 for the Japanese
population (Figures 3b-3d). These combinations, however, may changewith various COVID-19 strains that could exist in other populations andfurther investigation is necessary to identify the optimum combinationof primers for the Japanese population as cross-border travel brings
different COVID-19 strains that react to different primers.

Sample observation of RT-PCR data of irregular samples
Below are several examples of unique and irregular samples and
its RT-PCR data that could serve as future hypothesis for identifying
and detecting the causes of false negative RT-PCR results.In the firstcase, samples reacted only to the CDC-N2 primer but not to the JPN
primers. One of the most problematic cases observed are the samples
whereby JPN-N2 primer resulted in negative albeit responding positive
to CDC-N2 or CDC-N1 primers. Since Japanese testing protocol for
COVID-19 is JPN-N2 alone, registered Japanese clinical laboratories
are expected to call based on N2 primer result. However, in instancessuch as these, the importance to provide additional information
regarding the test result for CDC is recognized and attending physicians
are strongly urged to retest the patient again via RT-PCR or utilize othertesting methods such as CT Scan and antigen. This would diminish therisk of misinformation and potentially spread of the infection to othersfor patients, whether symptomatic or asymptomatic.
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Citation: Tsutae W, Chaochaisit W, Aoshima H, Ida C, Miyakawa S, et al. (2021) Detecting and Isolating False Negatives of SARS-Cov-2 Primers and Probe Sets among
the Japanese Population: A Laboratory Testing Methodology and Study. J Infect Dis Ther S1:004.

Page 4 of 6

Among the 4 primer-probe sets, only CDC-N2 showed positive
signal at late cycle (40 x) for one case. This patient (male patient A)
became symptomatic 14 days before testing and upon taking PCR test
on day 5, tested positive. After 1 week of quarantine and the publichealth office has lifted his self-quarantine, he voluntarily retested toreconfirm his status. His results showed that his test result would be
negative under Japan NIID’s JPN-N2 standalone testing protocol, but
the results clearly identified that he is still positive on CDC-N2 andwould be declared positive if this person were to be tested at a laboratory
in the US, where CDC-N2 is the standard protocol.
The second case also reflects how JPN-N2 was negative while
CDC-N1 and CDC-N2 both showed positive responses at later cycles
than JPN-N2 positive control (36.54 cycles and 37.44 respectively).

Primers vs. viral genetic variations
In an effort to try to explore the primer(s) effectiveness in current
virus variations, an exploratory data analysis has been conducted over84 virus strains from Japan, retrieved from NCBI GenBank genetic
sequence database as of August 28, 2020.
From extracted Japan’s samples, a phylogeny tree was generatedusing tool from NCBI Virus [7]. The resulting tree shows different
clades connected in hierarchies, suggesting that there are different virus
mutations in the geographical region. Next, a sequence alignment using
primers nucleotide from different countries against all coronavirus 2
strains from Japan was performed, using Blastn. The alignment results
are then filtered to contain only perfect consecutive alignment of all
nucleotides, meaning identities must be exactly equal to the length ofprimer oligonucleotide sequence with no gap. With such a rigid filter,the number of alignment matches were visualized using iTOL [8] andmany virus strains those do not match with some primers are found as
shown in Figure 4.

Discussion
There are numerous factors that could affect RT-PCR result forthe detection of SARS-CoV-2 ranging from sample collection method,collection timing, virus inactivation technique, and many other RTPCR procedures. There are testing guidelines for COVID-19 test
in many countries including NIID in Japan, CDC in USA, China,
Germany and others. However, it is possible that results discrepancy
occurs due to primers efficiency, sufficient viral RNA present in thesamples, and due to genetic variation at the primer-probe bindingsites ever after following the testing protocols and guidelines strictly.
Given that Genesis Healthcare followed all protocols and guideline very
strictly for sampling process, sample handling, sample transportation,
preservation, RNA extraction and RT-PCR process. In order to avoid
experimental error and data quality, multiple checkpoints including
positive control was used. However, irregularities were observed most
likely due to the variation in primer efficiency and sensitivity (Figures 3a-3h) as well as might be due to occurring variation at the primer-probe
binding sites.
It’s hypothesized that various virus strains align differently todifferent primers used globally due to its unique sequence. Specifically,at the time of analysis, there are 15,745 SARS-CoV-2 sequences
submitted to NCBI GenBank [9] where each sequence contains different
combination of variants/mutations. Therefore, to further validate ourassumption, the virus mutation data and primers oligonucleotide
sequences were investigated (Figure 3).
The finding also coincides with the study [10] reporting that of 33
oligonucleotides developed by different centers and shared by WHO,
79% (26) has at least one genome mutation at the primer binding sites
from GISAID database containing 1,825 SARS-CoV-2 genomes.

Figure 3: The detectability of COVID-19 positive or negative cases differs depending upon the primer-probe sets used. a) A RT-PCR results
for JPN-N2 and JPN-N1 primer-probe sets: JPN-N2 showed relatively higher signal intensity compared to JPN-N1. b) Testing Protocol byGenesis Healthcare incorporating various primers including Japan’s NIID testing protocol (Primers: JPN-N2, CDC-N1 and CDC-N2). Test results
irregularities: c) A Japanese case that was detected negative for JPN-N2 and CDC-N1 however was detected positive for CDC-N2. d) A Japanesecase that was negative for JPN-N2 however was detected positive using CDC-N1 and CDC-N2 primer-probe sets. e) A Japanese case that was
detected negative for JPN-N2 and CDC-N2 while showed positive for both CDC-N1 primer-probe sets. f) A Japanese case that was positive for
JPN-N2 however was detected negative by using CDC-N1 and CDC-N2 primer-probe sets. g) A Japanese case that was detected positive for
CDC-N2 and week positive (low signal intensity) for JPN-N2 and negative for CDC-N1 primer-probe sets. h) A Japanese case that was detected
positive for both JPN-N2 and CDC-N1 but negative for CDC-N2 primer-probe sets.

J Infect Dis Ther, an open access journal
ISSN: 2332-0877

Volume S1 • 1000004

Citation: Tsutae W, Chaochaisit W, Aoshima H, Ida C, Miyakawa S, et al. (2021) Detecting and Isolating False Negatives of SARS-Cov-2 Primers and Probe Sets among
the Japanese Population: A Laboratory Testing Methodology and Study. J Infect Dis Ther S1:004.

Page 5 of 6

Figure 4: Primers and probes locations were identified from 84 SARS-CoV-2 genome sequences from Japanese COVID-19 samples where
mutations were identified and false negatives will most likely occur.

While one dominant strain will test positive for a specific primerprobe set, there might be irregularities that will not display positive forother primer-probe sets, most likely resulting in false negative calls.
In this respect, laboratories should examine their primers and probes
against all known viral genome sequences to ensure that the selectedprimers will not result in inaccurate calling. In addition, different
combination of primer-probe sets should be used, at least three or four
primer-probe sets are recommended by Genesis Healthcare to reduceor overcome false negative results constraint by RT-PCR detection
method.

Conclusion
The conflicting positive/negative results from the same samples that
occur among different primers (JPN-N2, JPN-N1, CDC-N2, CDC-N1)
outline 1) the possibility of high occurrences of false negative testing
results by laboratories where JPN-N2 is the only primer used for SARSCoV-2, and 2) the probability that false negative/positive will likely
occur when the primer binding site lies in the virus mutated location
for JPN-N2 and CDC-N2.

economic growth, this may also lead to the introduction of new strains
to different populations furthering the demand for SUP.

Acknowledgement
We acknowledge the physicians from the originating medical
facilities responsible for obtaining the specimen from patients.

Conflict of Interests
The authors declare that there is no conflict of interest.

Ethics Statement
Biological samples were collected from volunteers, selfmedication testing participants and patients under physician careadministering clinical testing for SARS-CoV-2 (COVID-19). Samples
were anonymized and de-identified, so laboratory technicians andresearchers were blind to the identity of the patients. Only secondary
non-identifying data including age, biological sex and symptoms
were provided. All experimental protocols were approved by Genesis
Healthcare’s Ethics Committee.

This initial observation also suggests testing CDC-N2 in addition
to JPN-2 primer, especially for symptomatic patients, with the objective
to reduce false negatives and increase accuracy. Lastly, the inclusivityof CDC-N1 will result further in reducing false negatives but using
CDC-N1 alone may also result in false positive calling and therefore
should not be used without either JPN-N2 or CDC-N2.

Data Availability

Further investigation by sequencing these irregular samples will
identify which primers react to different strains of the virus existing
among different populations. Secondly, these irregularities raise theneed for the international testing community to explore a standardized
universal primer-probe sets combination for more accurate RT-PCRbased COVID-19 testing, which shall help to detect different virus
strain’s reaction to selected primers by each country’s testing protocol.
This is a critical consideration to ensure accurate diagnostic testing
results entailing public health and safety. While more countries are reopening their borders and resuming international travel to sustain theThis work is supported by Genesis Healthcare Corporation. The
sponsor has no influence in the study other than providing resources
and facility in conducting research and publication.

J Infect Dis Ther, an open access journal
ISSN: 2332-0877

The data used and produced in this research are available at:https://github.com/genesis-healthcare/covid-19_pcr_irregularities

Funding Sources

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