厚生連医誌

第31巻1号

40〜41

2022

Brief report

Quality control of severe acute respiratory syndrome
coronavirus 2（SARS-CoV-2）polymerase chain reaction（PCR）test for coronavirus disease 2019（COVID-19）
through our cross-contamination experience
Section of genetic analysis, Department of pathology, Nagaoka Central Hospital;
Medical specialist in Laboratory medicine and Pathology1）, Clinical technologist2）

Toshihiko Ikarashi1）, Hisayoshi Kosugi2）
Our pathological genetic laboratory has been established in 1999, and performed coronavirus PCR testing fromApril, 2020. The genetic laboratory（Biosafety level 2）isisolated room of air volume 7.5×5.5m×3m. We usedchemical reagents: SARS-CoV-2 Direct Detection RT-qPCR
Kit®（Takara, RC300A）, Positive Control RNA（US N1/N2, Takara, RC351A）. Realtime PCR was done with thePCR device AriaMx®（Agilent）. Coronavirus PCR test is
performed on the basis of our standard operating procedure（ISO15189: 2012, SOP. bacteria and others-401, 3rd ed,2020/12/07）.
We recognized the PCR contamination in August,2021, and our accredited laboratory was required to establish a corrective action on the basis of ISO15189; 2012.
The causes of our contamination were analyzed
based on a guidance of PCR procedure and a pursuit procedure of causes presented by the reagent-manufacturing
institute（Table 1）. The pollution seemed to occur mainlyby the incomplete isolation between a chemical reagent
adjustment area and a positive control/the specimen mixture area（non-conformity: 5.2.6 facility maintenance andenvironmental conditions in ISO15189;2012）, delicate
procedure before and after opening of tube and 8-well PCR
tube（5.3.1.3 equipment instructions for use）, contact pollution of tube cap（5.3.1.3）, incomplete 3S procedure ofappliance and pollution site. 3 months were required tosuppress the contamination（5.2.6）. The cooperation ofTakara Institute, Agilent, and AS ONE, was a great help toremove the pollution.
We can establish our prophylactic check list against
PCR contamination as follows: creation of a check list
（Table 2）and review of the SOP（standard of procedure）
in manual, complete separation of the working areas: achemical reagent adjustment area and a positive control/thesample mixture area, no share appliances between working
areas, each working place was locked with new safetycabinet, centrifuge the tube before open and spin the intratubular droplets down, smear test of instrument/the pollution site with the distilled water, use the micropipette tip
with a ﬁlter, wipe with hypochlorous acid, ultraviolet irra-diation, and an education of PCR procedures. This education included the proper handling the micropipette not tocontact any intra-cabinet equipment, no instant opening of
any cap to reduce splashing pollution, spin tube and 8-well
PCR tube down before open their caps, keep out the innercap surface from pollution by no direct touch and placing
on a torn aluminum-sheet rest for temporary installation,
avoid hanging the injection tip filled with positive control
or sample over or around 8-well PCR tubes, avoid opening
the 8-well PCR tubes after PCR not to pollute by PCR
products, and push into a plastic bag and take out fromgenetic laboratory. This check list will satisfy the requirements of training（5. 1. 5 in IS015189）and facility maintenance and environmental conditions（5. 2. 6）, and help
the revision of Standard Operating Procedures（SOP）ofSARS-CoV-2 PCR test.
Key words : SARS-CoV-2, COVID-19, PCR, contaminationin laboratory, ISO15189; 2012, corrective action
和文抄録短報長岡中央病院病理部遺伝子検査室における新型コロナウイルス（SARS-CoV-2）PCR 検査のコンタミネーションの経験
長岡中央病院 病理部 遺伝子検査室；臨床検査専門医、病理専門医1）、臨床検査技師2）
五十嵐俊彦1）、小杉久良2）

2021年 8 月に遺伝子検査室における新型コロナウイルス（SARS-CoV-2）PCR 検査のコンタミネーションを経験した。試薬調整作業台と陽性対照・検体混入作
業台の区分が不完全でクロスコンタミネーションを来したこと、手技の不手際、施設・装置・備品の3S 不良等が原因と考えられた。ISO15189; 2012是正措置要求－ 40 －

Quality control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction(PCR) test for coronavirus disease 2019 (COVID-19) through our cross-contamination experienceに基づく対処により、 3 ヶ月後に現状に復帰できた。
この是正措置作業を通して、日常業務に有効なチュックリストを作成した。関連施設の指導に感謝し、品質マネジメントシステムに基づく対処の有効性が確認された。

キーワード : SARS-CoV-2、COVID-19、PCR、検査室
内でのコンタミネーション、ISO15189; 2012、
是正措置

Table 1. Measures for the safe inspection to remove contaminating threats
1 ．conﬁrmation of the risk by the check list
2 ．detection of the pollution part by the smear test, negative control reacts with an unopened kit（more than N＝8 ）, consideration of contamination in procedure step（sampling, pretreatment, reagent adjustment, addition of positive control orsample, PCR）
3 ．isolation of each working space
4 ．decontamination of the laboratory by wipe with sodium hypochlorite and ultraviolet irradiation before and after inspection
5 ．lay aluminum foil on working area to assume it a clean site
6 ．changing expendable supplies to a new article
Table 2. Check list for contamination measures
1. in general
1 -1. divide working areas separately into a pretreatment area of the specimen, a reagent adjustment area, and a positivecontrol or specimen addition area
1 -2. do the operation in a safety cabinet
1 -3. prepare exclusive equipment such as the centrifuge, microtube, micropipette, and hydrophobic filter-tip, and do notshare it in other area
1 -4. use a hydrophobic ﬁlter-tip tip
1 -5. wipe every equipment and tool with a sodium hypochlorite solution before and after inspection
1 -6. lay aluminum foil on working area to assume it a clean site
1 -7 clean a laboratory once a week
1 -8. irradiate ultraviolet if necessary
2. at pretreatment
2 -1. wear the white robe, preventing the contiguity pollution by the sleeve, gloves, cap, and mask for exclusive use aroundthis area,
2 -2. wipe every equipment and tool with a sodium hypochlorite solution before and after inspection
2 -3. lay aluminum foil on working area to assume it a clean site
2 -4. do not touch the backside of the cover at opening a tube and 8-well PCR（polymerase chain reaction）tubes
2 -5. cover tube immediately to decrease volatilization
2 -6. seal up the waste in bag immediately and discard
3. at chemical reagent adjustment area, mix master ﬂuid and dispensation
3 -1. wear the white robe, preventing the contiguity pollution by the sleeve, gloves, cap, and mask for exclusive use aroundthis area
3 -2. do not bring samples and the positive control into the adjustment area to prevent contamination
3 -3. wipe every equipment and tool with a sodium hypochlorite solution before and after inspection
3 -4. lay aluminum foil on working area to assume it a clean site
3 -5. warn a micropipette tip touching at a safety cabinet to avoid any pollution
3 -6. do not touch the backside of the cover at opening a tube and 8-well PCR tubes
3 -7. seal up the waste in bag immediately and discard
4. at sample or positive-control addition area, and PCR area
4 -1. wear the white robe, preventing the contiguity pollution by the sleeve, gloves, cap, and mask for exclusive use aroundthis area
4 -2. do not bring samples and the positive control into the adjustment area to prevent contamination
4 -3. wipe every equipment and tool with a sodium hypochlorite solution before and after inspection
4 -4. lay aluminum foil on working area to assume it a clean site
4 -5. warn a micropipette tip touching at a safety cabinet to avoid any pollution
4 -6. spin-down with centrifuge before opening a tube and 8-well PCR tubes
4 -7. do not touch the backside of the cover at opening a tube and 8-well PCR tubes
4 -8. avoid adding sample over other sample tube to avoid any pollution
4 -9. do not open 8-well PCR tubes after PCR
4 -10. seal up the waste and PCR products in bag immediately and discard－ 41 －