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PCR 技术介绍 Introduction to PCR Technology PowerPoint PPT Presentation


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PCR 技术介绍 Introduction to PCR Technology. 济南艾迪康医学检验中心有限公司 PCR 实验室 杨超. 主要内容. 一. DNA 结构和复制 二. PCR 1. PCR 发展简史 2. PCR 基本概念 3. PCR 原理 三. 荧光定量 PCR 检测 HBV 1. 荧光定量 PCR 原理 2. 荧光定量 PCR 工作流程 3. 荧光定量 PCR 结果分析 4. 荧光定量 PCR 应用 四.基因芯片技术平台

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PCR 技术介绍 Introduction to PCR Technology

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PCRIntroduction to PCR Technology

PCR


. DNA

. PCR

1. PCR

2. PCR

3. PCR

. PCRHBV

1. PCR 2. PCR

3. PCR 4. PCR

.

HPV23


DNA

DNA


O-

O-

5

5

O=P

O=P

OCH2

OCH2

C

T

O

O

O-

O-

3

3

OH

OH

H

H

O

5

O=P

OCH2

C

O

O-

3

OH

H

A

G

C

T


DNA

DNA


1.DNA

2.RNA


3.DNA


  • 1985PE-CetusMullisPCR

  • DNA

  • E-coli DNAPCR

  • DNAPCRPE-CetusPCR

  • 1993Mullis


PCR

(Polymerase Chain Reaction)


PCR

DNA Mg2+


DNA


95

55

72

Taq

2

1

Taq


1

2


DNA

DNA

TaqDNA

dNTP

Mg2+


MJ Research, Inc.

PTC-100

PTC-150

PTC-200

PTC-220

PTC-225


ABS, Inc.

PE-2400

PE-2700

PE-9600

PE-9700


PCR

1

2.

3.

4.


1

2

3

94

72

55

22

1

2

3

4

5

min

PCR

1~3

25~30

DNA

2

DNA

100

DNA

DNA

DNA



PCRPCRPCR

PCR (Real-time PCR)

PCRPCRPCR


PCR

1. TaqMan

2. LightCycler

3.


TaqMan


LightCycler


P

C

R





HBV EDTA200ul

TB

HPV

CT




250C4DEPCRNAase


  • DNA

  • RNARNARNA1:45mol/L GITC()RNA


  • DNAEDTA

  • RNA GITC

  • RNA


  • /-70

  • DNA10mmol/L Tris

  • 1 mmol/L EDTA(pH 7.5-8.0)4

  • RNA-80

  • -20

  • GITCRNA7




  • 70%




(HBV)

  • 1.0 103copies/ml HBV DNA 4.2 109copies/ml

  • 2. HBVDNA> 4.2 109copies/ml> 4.2 109copies/ml

  • < 1.0 103copies/ml< 1.0 copies/ml

  • 6.666E+066.666 106 copies/ml


PCR

1.

2. mRNA

3. SNP


HBV DNA



HBV DNA


  • HBV




YMDD

YMDD:

YIDD

YVDD



DNA

PCR

PCR

PCR


DNA

1:

2:


/

DNA/RNA

PCR/RT-PCR


DNAPCR

Biotin

Primer up

Primer down

Biotin

PCR



Human Papillomaviruses, HPV

DNA

  • DNA8Kb

  • 5055nm


    • 1004030

    • HPV

    • 1HPV 61142

    • 2HPV 16 18313335394551525356585966 68


HPV

  • Regoni Stern

  • 1977LavertyHuman papillomavirusHPV

  • 89.Keerti ShahHPV

  • 1995IARCHPV


HPV

HPV

  • HPV95%

  • HPV1.55

  • CIN3HPV99.7


HPV

  • HPV E6p53

  • HPV E7RBRB E2F

  • HPV16-E5CDKp27


HPV

58

HPV HPV

HPV


  • HPVHPV

  • PCR18

    5HPV


HPV3335

HPV1643


HPV

HPV18

Primer up

HPV16

HPV58

Primer down

HPV1643


HPV


HPV


505%80%13.1528.8%

7010.28/10903.25/10


4580

354.8200034.1



100DNA9035301137HPV 1HPV 611424344 2 HPV 16 18 31 3335 3945 51 52 5356 5859 66687383MM43HPV45HPV3959HPV5258

HPV1651HPV185639


HPV


HPV33

Other

HPV31

HPV45

HPV16

HPV18

HPV

  • HPV611

  • HPV161831334558

HPV


HPV

1.1153119

/ 100

226

2. HPV


2001TBSThe Bethesda System)

ASCUSAtypical Squamous Cells)

ASC-US:

ASC-H

L-SILHPVCIN I

H-SILCIN IICIN III

SCC

AGC Atypical Glandular Cells)

AISAdenocarcinoma In Situ)

CS

CINCervical Intraepithelial Neoplasm)

CIN I

CIN II

CINIII


80

8

/HPV

<30

HPV

HPV

CIN I

415

>30

CIN II/III

6~24

510

HPV

HPV


CINCC

CIN


HPV5-10

2123HPV5-870103HPV3

25-3065


13


  • 50-70

  • LCT TCT

    1HPV

  • 2HPV

  • HPV DNAPCR PCR

    (98%)


CCRF

pap smear+HPV

TCT+HPV


  • HPV


HPV35

HPV

HPV


CIN 909510

45

8

46HPV


21

30

1

HIV


3

24



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