厚生連医誌

第31巻1号

34〜35

2022

Brief report

Interpretation of borderline results with 40<Cq≦45 in
real-time one-step reverse transcriptase-polymerase chain
reaction（RT-PCR）for severe acute respiratory syndrome
coronavirus 2（SARS-CoV-2）of coronavirus disease 2019
（COVID-19）used nasopharyngeal scraped specimens todecrease its false negativity
Section of Genetic analysis, Department of Pathology, JA Niigata Nagaoka Central General HospitalToshihiko Ikarashi
Background : We have diagnosed SARS-CoV-2 of COVID19 by one-step RT-PCR with TaqMan probe tonasopharyngeal scraped specimens according to thestandard of procedure（SOP）established by National
institute of infectious diseases in 2020. Its sensitivityof lowest level was required at viral RNA 5 copies/μl
by 45 times of PCR and, furthermore, its standard
positive value was threshold cycle （Cq）≦40（50copies/μl≦）. But the increasing proliferation
curve after 40 < Cq should have reviewed to ruleout false negative cases. In this report we examined
the number of Cq in lower viral RNA copy.
Materials and methods : The positive RNA of SARS-CoV-2
was obtained from Nihon gene research laboratories.
The standard curve was examined according to RNA
level from 2.5/μl to 50000/μl. Viral RNA waschecked by 45 times of PCR with one-step RT-PCRwith TaqMan probe（Takara SARS-CoV-2 direct
detection kit）and AriaMX Real-Time PCR（Agilent）. The relationship between viral RNA level andCq value was checked.
Results : At RNA 5 copies/μl, Cq was 35.5（mean）±0.745（σ）, and at RNA 2.5 copies/μl, Cq 36.6 ±1.52. On semi-logarithm graph, there were 2.1copies/μl and 3.8 copies/μl, at 5% hazard ratio and
0.3% hazard ratio, respectively.
Conclusion : In the false-positive case unsatisﬁed the standardized diagnostic criteria of Cq≦40 in PCR test,
we found that 2-4 viruses were present after 40
times of PCR cycles（40 < Cq）. Supposing that the
doubling time of virus is eight hours, the virus shallgrow up to 128-256 copies/μl after 2 days, when
the PCR test shall turn to positive. Therefore, thefalse negative case with 40 < Cq≦45 should be
intensely followed with re-examination to prevent a
possibility of the nosocomial infection by patientswith false-negative judgment. The above-mentioned
advice shall be surely recognized by an addendum

report of the ampliﬁcation plot ﬁgure demonstratinglate increasing plot curve pattern.
Key words : one-step reverse transcriptase-polymerase chain
reaction（RT-PCR）, AriaMX（Agilent）, real-time
PCR, severe acute respiratory syndrome coronavirus2（SARS-CoV-2）, coronavirus disease 2019（COVID19）, nasopharyngeal scraped specimens, decreasing
false negativity, 40 < Cq≦45
和文抄録

短報
コロナウイルス病2019（COVID-19）の重症急性呼吸症
候群コロナウイルス 2（SARS-CoV-2）診断のための鼻咽頭擦過標本を使用したリアルタイム・ワンステップ
逆転写酵素−ポリメラーゼ連鎖反応（RT-PCR）において、偽陰性を減少する為の40＜Cq≦45症例の対応
JA 新潟長岡中央綜合病院、病理部、遺伝子検査室
五十嵐俊彦
コロナウイルス病2019（COVID-19）の重篤急性呼吸症候群コロナウイルス 2（SARS-CoV-2）診断のための鼻咽頭擦過標本を使用したリアルタイム・ワンステップ逆転写酵素 - ポリメラーゼ連鎖反応（RT-PCR）において、保険収載の判定基準 Cq≦40（注意： Cq＝Ct、増殖曲線と閾値の交点での PCR サイクル数）を満たせない40＜Cq≦45の陰性症例を単純に陰性として放置すべきではない。検量線より、40＜Cq においてウイルスが少数ながら認められることより、初回検査 2 日以降の再検査が望ましく、診断医への警告として増殖曲線図を追加添付することが有用である。
キーワード：リアルタイム・ワンステップ逆転写酵－ 34 －Interpretation of borderline results with 40<Cq≦45 in real-time one-step reverse transcriptase-polymerase chain reaction (RT-PCR) for severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) of coronavirus disease 2019 (COVID-19) used nasopharyngeal scraped specimens to decrease its false negativity

素−ポリメラーゼ連鎖反応（RT-PCR）、AriaMX（アジレント）、コロナウイルス病2019（COVID19）、重症急性呼吸症候群コロナウイルス 2－ 35 －（SARS-CoV-2）、鼻咽頭擦過標本、偽陰性例の抑制、40<Cq≦45