HPV原材料探針基因的選定實驗

1、Determine probes genes8. 探針基因的確定8.1 Basic Data of Test8.1基本測試數據Document No.文件號BMT-TF-SFDA-01-R0Test Name實驗名稱The test of determining probes genes探針基因的選定實驗Purpose of the Test實驗目的To determine probes genes of BMT HPV Genotyping 9G membrane KIT確定BMT HPV 分型 9G膜試劑盒所需的探針基因 Tester試驗操作人 (sign)（簽名）Confirmor確認人

2、 (sign)（簽名）Date of Start (MM/DD/YY)開始日期（年年/月月/日日）Date of End(MM/DD/YY)結束日期（年年/月月/日日）Test Site測試地點 BMT 2-2-2 (Temperature : ,Humidity: ) （溫度： ，濕度 ）Used Material/Specimen 使用的材料/標本Using a sample produced by diluting DNA HPV 16, 18, 31, 33 from 1.0 x107 copies up to 3 copies1) 通過將HPV 16, 18, 31, 33DNA從1

3、.0 x107拷貝稀釋到3拷貝來獲得標本2) Using a sample produced by diluting DNA HPV 45, 26, 35, 39, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, 73, 6, 10, 11, 32, 34, 40, 42, 43, 44, 55, 62, 71, 74, 81 from 1.0 x107 copies up to 1.0 x10 copies2）通過將HPV 45, 26, 35, 39, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, 73, 6,10, 11,

4、 32, 34, 40, 42, 43, 44, 55, 62, 71, 74, 81, 52 DNA從1.0 x107拷貝稀釋到1.0 x10拷貝來獲得標本Product No.產品編號Product Name產品名稱QC#BMT HPV1 Genotyping 9G Membrane StripBMT HPV1 分型9G膜試紙條BMT HPV2 Genotyping 9G Membrane StripBMT HPV2 分型9G膜試紙條Hybridization Buffer 雜交緩沖液HC Cy5-DNA Washing Buffer沖洗緩沖液10mM Tris buffer (pH8.0

5、)10mM Tris 緩沖液(pH為8.0)PCR premixPCR預混合物BMT HPV Primer setBMT HPV引物組Name of Folder to Save Original File保存原始文件的文件夾名稱 Determine probes genes Determine probes genes 探針基因的確定探針基因的確定 Name of folder where the original file is saved 保存原始文件的文件夾名稱 Name of original file 原始文件的名稱8.2 Test Method8.2 實驗方法Preparatio

6、n1) 準備1-1) Preparing Materials1-1) 物品準備The DNA used for test was used within the set effective period by producing as fixed concentration after quantifying with Nanodrop usingHPV L1 DNA (Human Papilloma Virus L1 DNA, Code 08/023) which is the national standard product parceled out by the Korean Food

7、 and Drug Administration. The used samples are as follows.在設定的有效期內使用已經過Nanodrop定量檢測的確定濃度的DNA作為測試標本。 該測試樣本是由韓國食品與藥品管理局分配的國家標準品HPV L1 DNA（人乳頭瘤病毒L1 DNA, 代碼08/023）。使用的標本見下表。Used Material/Specimen使用的材料/標本Sample : HPV L1 DNA (Human Papilloma Virus L1 DNA, Code 08/023)樣本：HPV L1 DNA（人類乳狀瘤病毒L1 DNA，編碼08/023）H

8、PV typeHPV亞型Management No.、管理號HPV typeHPV亞型Management No.管理號HPV 16HPV-16-KFDAHPV 06HPV-06-KFDAHPV 18HPV-18-KFDAHPV 10HPV-10-KFDAHPV 31HPV-31-KFDAHPV 11HPV-11-KFDAHPV 33HPV-33-KFDAHPV 32HPV-32-KFDAHPV 45HPV-45-KFDAHPV 34HPV-34-KFDAHPV 26HPV-26-KFDAHPV 40HPV-40-KFDAHPV35HPV-35-KFDAHPV 42HPV-42-KFDAHPV

9、 39HPV-39-KFDAHPV 43HPV-43-KFDAHPV 51HPV-51-KFDAHPV 44HPV-44-KFDAHPV 52HPV-52-KFDAHPV 54HPV-54-KFDAHPV 53HPV-53-KFDAHPV 55HPV-55-KFDAHPV 56HPV-56-KFDAHPV 62HPV-62-KFDAHPV 58HPV-58-KFDAHPV 71HPV-71-KFDAHPV 59HPV-59-KFDAHPV 74HPV-74-KFDAHPV 66HPV-66-KFDAHPV 81HPV-81-KFDAHPV 68HPV-68-KFDAHPV 69HPV-69-K

10、FDAHPV 70HPV-70-KFDAHPV 73HPV-73-KFDA1-2) The strip for determining probes1-2) 用于確定探針的試紙條The strips used in this test for determining probes were manufactured according to the production procedure of BMT HPV Genotyping 9G membrane KIT in Chapter 1 of Manufacturing Process. The strips were within the

11、 warranty period during the test. The optional probes immobilized on the test chip includes 17mer of 34 HPV types.本次實驗所用試紙條是按照BMT HPV 分型 9G膜試劑盒制造流程中第一章規(guī)定的生產步驟來制作的。實驗時試紙條仍在有效期內。固定在測試芯片上的可選探針包括長度為17mer 的34種HPV亞型。The genes of 17mer of 34 HPV probes were chosen from 6725-6741region of Target DNA HPV 16.

12、 The probes were immobilized on the membrane to make test strip.17mer 34種HPV探針基因選自目標DNA HPV 16 的6725-6741區(qū)。將探針固定在膜之上制成實驗試紙條。1-3) Sequence of probes genes1-3) 探針基因序列HPV probeHPV探針Sequence(5-3)序列(5-3)1HPVS 16GGGGGGGGG TTTTTTTTT GTA CCT ACG ACA AGG GGA GG 2HPVS 18GGGGGGGGG TTTTTTTTT GTA TAG CAG ACT TGT

13、 TGA GG3HPVS 45GGGGGGGGG TTTTTTTTT GTA TAG TAG ACA AGT GGA GG4HPVS 31GGGGGGGGG TTTTTTTTT GTA TTT AAG ACA AGG TGA GG5HPVS 33GGGGGGGGG TTTTTTTTT ATA TAT AAG ACA AGT TGA AG 6HPVS 52GGG GGG GGG CTT TAT CAT ATA CCT TCG TCA TGG CGA GG7HPVS 66GGG GGG GGG CTT TAT CAT ATA CCT TCG CCA AGT GGA GG 8HPVS 56GGG G

14、GG GGG CTT TAT CAT GTA CCT TAG ACA AGT GGA GG9HPVS 59GGG GGG GGG CTT TAT CAT ATA TGC CAG ACA AGT GGA GG 10HPVS 35GGG GGG GGG CTT TAT CAT ATA TTT AAG GCA TGG TGA AG11HPVS 58GGG GGG GGG CTT TAT CAT ATA TGT ACG TCA TGT TGA AG12HPVS 69GGG GGG GGG CTT TAT CAT GTA TAT AAG GCA TGG TGA GG 13HPVS 51GGG GGG G

15、GG CTT TAT CAT ATA TAT TAG GCA TGG GGA AG 14HPVS 68GGG GGG GGG CTT TAT CAT ATA TGT TAG GCA AGT TGA GG15HPVS 39GGG GGG GGG CTT TAT CAT ATA TAC CAG GCA CGT GGA GG 16HPVS 70GGG GGG GGG CTT TAT CAT ATA TAC TAG GCA AGT GGA GG 17HPVS 26GGG GGG GGG CTT TAT CAT ATT TAT AAG ACA TGG CGA AG18HPVS 53GGG GGG GGG

16、 CTT TAT CAT ATA TGT TAG ACA AGC AGA GG19HPVS 73GGG GGG GGG CTT TAT CAT ATA TTT AAG ACA AGC AGA AG 20HPVSPCR GGG GGG GGG CTT TAT CTT GAC ATG KKG ARG ART ATG A21HPVSPC GGG GGG GGG TGA TTT ACA GTT TAT DTT TC22HPVSHC GGG GGG GGG TTT CCT AGT GGC TCT ATG GTA ACGGG GGG GGG part used for immobilization was

17、 ignored.忽略用于固定的GGG GGG GGG部分 1-4) Maps of strips immobilized with probes1-4) 固定了探針的試紙條的圖片Test strips immobilized with probes were specially manufactured according to the production procedure of BMT HPV Genotyping 9G membrane KIT in Chapter 1 of Manufacturing Process.固定有探針的實驗試紙條是按照BMT HPV 分型 9G膜試劑盒生

18、產流程中第一章規(guī)定的生產步驟來制作的。 The chips maps are as follows.芯片的圖片如下：BMT HPV1 Genotyping 9G membraneBMT HPV1 分型 9G膜BMT HPV2 Genotyping 9G membraneBMT HPV2 分型 9G膜- Genotyping types- 可分型亞型 HPV type A(52,66), B(56,59),C(35,51,58,68,69),D(39,70), and E(26,53,73) (14 types ) HPV A類（52，66），B類（56,59），C類(35,51,58,68,6

19、9)，D類(39,70)以及E類(26,53,73) (共14 種 )- Screening types- 可篩查類型Low risk HPV type 03, 06, 11, 27, 32, 40, 42, 43, 57 and 67 (10 types)低危HPV 03, 06, 11, 27, 32, 40, 42, 43, 57 和67型（共10種）Test Process2) 實驗過程The test of determining probes genes was performed as follows.探針基因確定實驗操作如下:After preparing a sample p

20、roduced by diluting DNA HPV 16, 18, 31, 33 form 1.0 x107 copies up to 3 copies and diluting DNA HPV 45, 26, 35, 39, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, 73, 6,10, 11, 32, 34, 40, 42, 43, 44, 55, 62, 71, 74, 81, 52 from 1.0 x107 copies up to 1.0 x10 copies a) 先將HPV 16, 18, 31, 33型DNA從濃度1.0 x107 拷貝

21、稀釋至3拷貝，將HPV45, 26, 35, 39, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, 73, 6, 10, 11, 32, 34, 40, 42, 43, 44, 55, 62, 71, 74, 81,52型DNA從濃度1.0 x107 拷貝稀釋至1.0 x10拷貝，用作實驗樣本This is PCR amplified according to the PCR Method (2-2) of Users Manual using this as template DNA.b) 使用上述樣本作為模板DNA，根據用戶手冊中PCR法(2-2)進行PC

22、R擴增。After that, cleanse according to the Washing Method (2-4) after performing hybridization in the strip according to the Hybridization Method (2-3).c) 之后，先按照雜交法(2-3)在試紙條上完成雜交，再根據沖洗方法(2-4)沖洗試紙條。Analyze the result by scanning the strip with BMT 1D scannerTM d) 使用BMT 1D 掃描儀TM 掃描試紙條并分析結果。When HPV test

23、s show “HPV Positive”, load the remaining of the 1st hybridization solution (110ul) on the “sample port” of HPV2 Strip (optional) for further genotyping results.e) 當HPV測試顯示”HPV Positive“時，將剩余的第一次的雜交緩沖液（110ul）加至HPV2 試紙條 (可選)上的“樣本端口”做進一步的分型檢測。HPV Positive: PCR above 300 and HPV 16, 18, 45, 31, 33 less

24、 than 300.HPV 陽性：PCR為300以上且HPV 16, 18, 45, 31, 33低于300.8.3 Conclusion and Decision8.3 結論與判定The results showed that only the HPV type same with standard material showed positive and the other HPV types all showed negative.結果表明只有與標準品一致的HPV類型才會顯示陽性，其他的HPV亞型均顯陰性。Selected probes sequences are listed in f

25、ollowing table.選定的探針序列如下：HPV probeHPV探針Sequence(5-3)序列(5-3) 1HPVS 16GGGGGGGGG TTTTTTTTT GTA CCT ACG ACA AGG GGA GG 2HPVS 18GGGGGGGGG TTTTTTTTT GTA TAG CAG ACT TGT TGA GG3HPVS 45GGGGGGGGG TTTTTTTTT GTA TAG TAG ACA AGT GGA GG4HPVS 31GGGGGGGGG TTTTTTTTT GTA TTT AAG ACA AGG TGA GG5HPVS 33GGGGGGGGG TTTTT

26、TTTT ATA TAT AAG ACA AGT TGA AG 6HPVS 52GGG GGG GGG CTT TAT CAT ATA CCT TCG TCA TGG CGA GG7HPVS 66GGG GGG GGG CTT TAT CAT ATA CCT TCG CCA AGT GGA GG 8HPVS 56GGG GGG GGG CTT TAT CAT GTA CCT TAG ACA AGT GGA GG9HPVS 59GGG GGG GGG CTT TAT CAT ATA TGC CAG ACA AGT GGA GG 10HPVS 35GGG GGG GGG CTT TAT CAT ATA TTT AAG GCA TGG TGA AG11HPVS