病原体検出マニュアル 2019-nCoV Ver.2

1 1 2019-nCoV 2 2 17 HP 2019-nCoV 2 2 5 2 2 13 2 2 15 2019-nCoV open reading flame 1a (ORF1a) spike (S) 2-step RT-PCR TaqMan one-step RT-PCR 2019-nCoV 1 2019-nCoV ( ) HP 2 RNA QIAamp Viral RNA Mini Kit RNA 1) 2) QIAamp Viral RNA Mini Kit (QIAGEN ) Distilled water (Deionized, Sterile, autoclaved, DNase free, RNase free Cat No. 318-90105 ( DDW) RNA 2 1) 2+ personal protective equipment (PPE) 2) RNase PCR QIAamp Viral RNA Mini RNA 1) (1) (15 25 C) (2) Carrier RNA 1 g/ l Carrier RNA ( )310 g Buffer AVE 310 l 1 g/ l Carrier RNA -20 C 3 Buffer AVL 80 C (3) Buffer AVL/Carrier RNA 1 Buffer AVL 560 l Carrier RNA l Buffer AV L / C a r r i e r R N A ( Handbook Table1.

2 2 8 C 80 C 5 Buffer AV L L /Carrier RNA 560 l -20 C (4) Buffer AW1 Buffer AW2 Buffer AW1 (Kit ) 96 100% 25ml Buffer AW2 (Kit ) 96 100% 30ml 2) (1) Buffer AVL/Carrier RNA 560 l (2) 140 l Buffer 15 vortex (15 25 C) 10 ( ) (3) (96 100%)560 l 15 vortex (4) (3) 630 l QIAamp (2ml ) 6,000 g (8,000 rpm) 1 QIAamp 2ml 3 (3) 630 l ( 2 ) (5) QIAamp Buffer AW1 500 l 6,000 g (8,000 rpm) 1 QIAamp 2ml (6) QIAamp Buffer AW2 500 l 20,000 g (14,000 rpm) 3 (7) (7) QIAamp 2ml (20,000 g ) 1 (8) QIAamp QIAamp Buffer AV E 60 l 1 6,000 g (8,000 rpm) 1 RNA -80 C 3.

3 2-step RT-PCR 2019-nCoV SuperScript IV Reverse Transcriptase (Thermo) Quick Taq HS Dymix (To y o b o) PCR 1) PCR 1 2019-nCoV 2 4 2) SuperScript IV Reverse Transcriptase (RT) [Thermo, 18090010. 50, 200 (PrimeScript RT reagent Kit, Takara RR037A )] [Quick Taq HS Dymix To yobo DTM-101 (PerfectShot ex taq , Takara RR005A )] 2019-nCoV Oligo(dT)12 18 Primer (Thermo, 18418012 ) Random Hexamers (Thermo, N8080127 ) Recombinant RNase Inhibitor (Takara-Bio, 2313A ) PCR (Wizard SV Gel and PCR Clean-Up System, Promega A9281 ) DDW No. Name direction sequence (5' to 3') Expected size (bp) ORF1a 1st NIID_WH-1_F501 Sense TTCGGATGCTCGAACTGCACC 413 1st NIID_WH-1_R913 Antisense CTTTACCAGCACGTGCTAGAAGG 2nd NIID_WH-1_F509 Sense CTCGAACTGCACCTCATGG 346 2nd NIID_WH-1_R854 Antisense CAGAAGTTGTTATCGACATAGC Seq NIID_WH-1_Seq_F519 Sense ACCTCATGGTCATGTTATGG Seq NIID_WH-1_Seq_R840 Antisense GACATAGCGAGTGTATGCC S 1st WuhanCoV-spk1-f Sense TTGGCAAAATTCAAGACTCACTTT 547 1st WuhanCoV-spk2-r Antisense TGTGGTTCATAAAAATTCCTTTGTG 2nd NIID_WH-1_F24381 Sense TCAAGACTCACTTTCTTCCAC 493 2nd NIID_WH-1_R24873 Antisense ATTTGAAACAAAGACACCTTCAC Seq NIID_WH-1_Seq_F24383 Sense AAGACTCACTTTCTTCCACAG Seq NIID_WH-1_Seq_R24865 Antisense CAAAGACACCTTCACGAGG 5 1st strand cDNA 1) ORF1a S 105/ l RNA 10 l DDW 990 l 2) 1) RNA 5 l DDW 10 l (5000 )

4 5000 3) RNA RNA 1 5 SSIV Buffer 5 l Random Hexamers 1 l Oligo(dT)12 18 Primer 1 l dNTP (2mM each) 5 l 100 mM DTT 1 l Recombinant RNase Inhibitor 1 l SuperScript IV Reverse Transcriptase 1 l RNA 10 l To t a l 25 l 4) 23 C 10 min 50 C 10 min 80 C 10 min 5) 35 l DDW PCR 1st PCR 6 1) 2 Quick Taq HS DyeMix 25 l Forward primer (50 M) l Reverse primer (50 M) l DDW l cDNA 5 l To t a l 50 l ORF1a NIID_WH-1_F501 NIID_WH-1_R913 S WuhanCoV-spk1-f WuhanCoV-spk2-r 2) DDW 5 l 3) PCR 94 C 1 min 94 C 30 sec 56 C 30 sec 68 C 1 min 68 C 5 min 2nd PCR 1) 2 Quick Taq HS DyeMix 25 l Forward primer (50 M) l Reverse primer (50 M) l DDW l 1st PCR 1 l To t a l 50 l ORF1a NIID_WH-1_F509 NIID_WH-1_R854 S NIID_WH-1_F24381 NIID_WH-1_R24873 2) DDW 1 l 1stPCR 1 l 3) PCR 94 C 1 min 94 C 30 sec 56 C 30 sec 40 40 7 68 C 1 min 68 C 5 min 4) 1stPCR 2ndPCR 5uL 2 ( ME, ) TAE ( ) ( ) 1stPCR 1st PCR 2 4 ORF a 1stPCR 292bp 2ndPCR 261bp S 1stPCR 329bp 2ndPCR 294bp 2019-nCoV nested PCR MarkerPositivespecimenControlMarkerPosit ivespecimenControl3002001004005003002001 00400500346bp261bp493bp294bp nested PCR PCR 8 5)

5 2nd PCR Nested RT-PCR 1 2019-nCoV 2 Nested RT-PCR 3 ( )PCR PCR Wizard SV Gel and PCR Clean-Up System Agencourt AMPure XP 1) PCR (45 l) PCR 45ul Membrane Binding Solution 2) SV minicolumn Collection tube 1 16,000 g 1 3) Collection tube SV minicolumn Membrane Wash Solution 700 l 16,000 g 1 4) Collection tube SV minicolumn Membrane Wash Solution 500 l 16,000 g 5 5) Collection tube SV minicolumn 16,000 g 1 6) SV minicolumn 30 l TE ( DDW) 1 16,000 g 1 ( ) ORF1a NIID_WH-1_Seq_F519 NIID_WH-1_Seq_R840 S NIID_WH-1_Seq_F24383 NIID_WH-1_Seq_R24865 Blast MN908947 ( 95 ) 2019-nCoV 2 9 ORF1a S 2019-nCoV 4.

6 TaqMan one-step RT-PCR 10 20 200 1000 l DDW 96well PCR 8 PCR TaqMan QuantiTect Probe RT-PCR Kit (QIAGEN Cat#204443) [AgPath-ID One-step RT-PCR Reagents (Thermo Cat# AM1005) TaqMan Fast Virus 1-Step Master Mix (Thermo Cat 4,444,432) ABI RT-PCR N Name Sequence (5 to 3 ) Position* Concentration N_Sarbeco_F1 CACATTGGCACCCGCAATC 28723-28741 600nM N_Sarbeco_R1 GAGGAACGAGAAGAGGCTTG 28850-28831 800nM N_Sarbeco_P1 FAM-ACTTCCTCAAGGAACAACATTGCCA-BHQ( QSY**) 28770-28794 200nM 128bp N (N2 ) Name Sequence (5 to 3 ) Position* Concentration NIID_2019-nCOV_N_F2 AAATTTTGGGGACCAGGAAC 29142-29161 500nM NIID_2019-nCOV_N_R2 TGGCAGCTGTGTAGGTCAAC** 29299-29280 700nM NIID_2019-nCOV_N_P2 FAM-ATGTCGCGCATTGGCATGGA-BHQ( QSY**) 29239-29258 200nM 158bp *:Position Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 **.]

7 TAMRA 10 ** Ve r3 RNA one-step RT-PCR(TaqMan ) QIAGEN QuantiTect Probe RT-PCR kit 1) RNA N N2 105/ l RNA 10 l DDW 90 l RNA50 l DDW450 l (103/ l) RNA (BamH I H5 ) 2) 1) 5 103/5 l 5 100/5 l 10 DDW450 l RNA50 l 1 3) RNA 1 RT-PCR N N2 2 Master mix l l Forward primer (10 M) l l Reverse primer (10 M) l l TaqMan probe (5 M) l l Quantitect RT mix l l DDW l l Template RNA 5 l 5 l To t a l 20 l 20 l mix` 30 11 4) 8 15 l 5uL 2 5) RNA 5 l 2 6) RNA 5 l 2 ( ) 7) PCR QIAGEN QuantiTect Probe RT-PCR Kit PCR Applied Biosystems Applied Biosystems 7500 Fast PCR Roche Diagnostics LightCycler 480 ( 480II 96) Applied Biosystems 7500 Fast PCR (Standard ) 50 C 30min.

8 95 C 15min. 95 C 15sec. 45 cycles 60 C 60sec.(Data Collection) LightCycler 480 II (480 96) Analysis Mode Cycle Temperature ( C) Time Ramp Rate ( C/sec) Acquisition Mode Assay Absolute Quantification (Standard Curve) Run Mode Standard 7500 Repoter FAM Quencher None 12 RT None 1 50* 30min. None Denature None 1 95 15min. None PCR Quantification 45 95 15sec. None 60 60sec. Single Cooling None 40 30sec. ** None **:Cooling Ramp Rate 8) 40 N N2 N2 2 N2 N 1 Ct Cq N2 N N2 2nd derivative RNA(Accession LC521925) RT-PCR N 7 N2 2 RNA LightMix Modular SARS and Wuhan CoV 518-499921 518-499914 E gene N gene SARS-like Master Mix Modular EAV RNA Extract.

9 Control 518-219963 LigthCycler Multiplex RNA Virus Master SARS-like 2003 SARS-CoV 2019-nCoV E gene N gene 2003 SARS-CoV 2019-nCoV SARS-CoV 2019-nCoV 13 ( ) 1 ver. 1 PBS(-) PBS(-) RNA RNA DNase RNA PBS(-) DTT 1.

10 DTT PBS(-) RNA *1 *1 2 200 1000 L 15mL 50mL PBS (-) dithiothreitol, DTT: Wako Cat#044-29221, 29223 PBS(-) ) 2019-nCoV DTT 1) PBS(-) 10% w/v DTT *2 10% DTT in PBS 2) 1 10%DTT in PBS 3) 15 4) RNA 2 DNase *2 10% DTT in PBS -20 C 3 PBS(-) 1) 1 3 PBS(-) 2) 1mL mL 2 mL 3) 20000 x g 30 4 C 4)