鹿亞超-數(shù)字PCR方法的建立及質(zhì)量指標(biāo)評(píng)估-PL-SV

1、數(shù)字PCR法的建及質(zhì)量指標(biāo)評(píng)估亞超/ Peter Lu上海靶向分醫(yī)學(xué)研究所20170914內(nèi)容分檢測(cè)法和質(zhì)量評(píng)估數(shù)字PCR技術(shù)特點(diǎn)和法建數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估實(shí)例分享保密 - 請(qǐng)勿外傳內(nèi)容分檢測(cè)法和質(zhì)量評(píng)估數(shù)字PCR技術(shù)特點(diǎn)和法建數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估實(shí)例分享保密 - 請(qǐng)勿外傳臨床分檢測(cè)技術(shù)檢測(cè)對(duì)象檢測(cè)技術(shù)基因突變(Gene mutation)DNA sequencing， ARMS，HRM拷貝數(shù)改變(Copy Number Variation, CNV)FISH, aCGHDNA sequencing， ARMS，HRM基因重排（Gene rearrangement)反轉(zhuǎn)錄+熒光定量PC

2、R（RT-qPCR)基因表達(dá)(mRNA/RNA expression） 蛋(質(zhì))表達(dá)(Protein expression)免疫組織化學(xué)（IHC）, ELISA DNA/RNA檢測(cè)技術(shù)媒介檢測(cè)熒光顯反應(yīng)靶序列Basic：堿效應(yīng)酸效應(yīng)DNA聚合酶 反轉(zhuǎn)錄酶基因變異基因修飾堿基配對(duì)酶內(nèi)切酶 外切酶熱變性復(fù)性/雜交核酸酶環(huán)化酶簡(jiǎn)單分類實(shí)時(shí)熒光定量PCR(Real-time PCR, qPCR)其它類型技術(shù)核酸序列檢測(cè)：代測(cè)序、代測(cè)序特定區(qū)域檢測(cè)：HPLC、基因表達(dá)芯、斑點(diǎn)雜交特定位點(diǎn)檢測(cè)：液態(tài)芯、SNP基因芯qPCR dPCRDigital DropletPCR (Biorad)3D dPCR(Li

3、feTech)RainDrop(RainDance)partitions/sampleVolume/reaction200001nL2000033nLLow1-10 millionpL levelLowThrough-putRatio of S/NMultiplexHighHighNoMediumNoMediumYesReal-time PCR, qPCR適合臨床檢測(cè)亞類繁雜TaqManMolecular Beacon PCR SURVEYORScorpion ARMSHS-ARMSADx ARMS PCR clampingPNA/LNA lockHRMCOLD-PCRCAST-PCRYIN-

4、YANG History of ARMSSource: from AmoyDxReal-time PCR, qPCR標(biāo)序列！熒光 PCR產(chǎn)物基因變異基因修飾實(shí)驗(yàn)設(shè)計(jì)法建難度：靶序列 基因重排 點(diǎn)-Germline 點(diǎn)-Somatic單重檢測(cè) 多重檢測(cè)臨床分檢測(cè)法質(zhì)量評(píng)估主要指標(biāo)靈敏度/敏感度 Sensitivity疾病組 對(duì)照組分析靈敏度 Analytical sensitivity臨床敏感度 Clinical sensitivity真陽(yáng)假陽(yáng)測(cè)試陽(yáng)性測(cè)試陰性(a)(c)假陰真陰(b)(d)檢出/定量限度 LOD/LOQ: Limit of Detection/Quantitation （95%

5、）特異性 Specicity分析特異性 Analytical specicity臨床特異性 Clinical specicity準(zhǔn)確度 Accuracy真實(shí)度 Trueness (25% SV)精密度 Precision (5-10% SV)重復(fù)性 Repeatability 重現(xiàn)性 Reproducibility線性范圍 Linear dynamic range擴(kuò)增效率 PCR eciencySource: ISO 5725檢出限術(shù)語(yǔ)使混亂：靈敏度 Sensitivity分析靈敏度 Analytical sensitivity最檢出限 Minimum detection limit功能靈敏度

6、 Functional sensitivity檢出限度 Limit of detection定量限度 Limit of quantitation內(nèi)容分檢測(cè)法和質(zhì)量評(píng)估數(shù)字PCR技術(shù)特點(diǎn)和法建數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估實(shí)例分享保密 - 請(qǐng)勿外傳技術(shù)特點(diǎn)絕對(duì)定量核酸分檢測(cè)Absolute quantication of nucleic acids 分液（20000.）稀有變異和序列檢測(cè)Rare Variant and sequences Detection絕對(duì)定量基因拷數(shù)變異檢測(cè)Gene copy number variation (CNV)Endpoint read 終點(diǎn)基因表達(dá)檢測(cè)Gene ex

7、pression兩通道 (FAM, VIC/HEX)Next-generation sequencing (NGS)單細(xì)胞分析Single cell analysis數(shù)據(jù)判讀式法建明確需求案設(shè)計(jì)oligo設(shè)計(jì)測(cè)試案qPCR亞類controls：檢測(cè)體系Internal control Reference samplesquantication calibratorPTCNTCBlanknegative extraction control結(jié)果解讀質(zhì)控品制備第2、3法物學(xué)證據(jù)體系建優(yōu)化臨床驗(yàn)證試劑耗材訂購(gòu)室內(nèi)體系建室間質(zhì)評(píng)標(biāo)準(zhǔn)流程質(zhì)控體系法建時(shí)的些考量Dx probe-FAM channel提信

8、噪(S/N)反應(yīng)條件，退溫度探針產(chǎn)商普通PCR擴(kuò)增儀反應(yīng)體系，Oligo濃度，酶，和PCR mastmixThreshold的確定Biomol Detect Quantif. 2016 Mar; 7: 920.內(nèi)容分檢測(cè)法和質(zhì)量評(píng)估數(shù)字PCR技術(shù)特點(diǎn)和法建數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估實(shí)例分享保密 - 請(qǐng)勿外傳質(zhì)量評(píng)估SelectivityFor dPCR, selectivity can be translated into the degree to which partitions classied aspositive contain one or more copies of the ta

9、rget sequence, and the negative partitionscontain no copy of the target sequence.NTC野臨床樣本：不同濃度陽(yáng)性臨床樣本，陽(yáng)性質(zhì)粒、spikein樣本質(zhì)量評(píng)估dynamic rangeFor each target fragment, a seven-point 10-fold calibration curve was volumetricallyprepared from 50 million to 50 copies per 50 L PCR reactionthe precision (and, there

10、fore, also the reliability) of measurement results is not constant acrossthis whole range due to stochastic eects which have an important inuence at the lower andupper limit of the range質(zhì)量評(píng)估Measurement precisionPrecision is a measure of the variability in independent measurement results obtained for

11、 thesame sample under stipulated conditions. Depending on the stipulated conditions,measurement precision can be divided into method repeatability, intermediate precision and reproducibility. Within the frame of a single labo- ratory validation, both the method repeatability andintermediate precisio

12、n need to be investigated. Repeatability is a parameter for the variabilityin results of measurements performed by a single analyst using the same equipment andreagents during a short period of time. Intermediate precision gives an estimate of the vari-ation in results from measurements made under c

13、onditions which are more variable thanrepeatability conditions. Ideally, the eect of all sources of variation that could occur duringroutine use in a single laboratory should be investigated. The assessment of methodreproducibility requires measurement results obtained by dierent laboratories. Thisi

14、nformation is quite valuable but not mandatory in case of a single laboratory methodvalidation 15.Anal Chem. 2016 February 16; 88(4): 21322139.質(zhì)量評(píng)估TruenessHorizon 圖Measurement trueness is an expression of how close the mean of an innite number (i.e. alarge number in reality) of results produced by t

15、he method comes to a reference value. Thereare three general approaches to obtain a suitable reference value:i) use of certied reference materials, ii) recovery experiments using spiked samples, and iii) comparison with results obtained from another method：qubit, ME_PCR, agilent2100,Beaming質(zhì)量評(píng)估Measu

16、rementuncertaintyMeasurement uncertainty may arise from many sources, and these sources of uncertainty canbe divided into ve dierent lev- els: random eects, run-to-run eects, laboratory bias, method bias and matrix variation eects 16. The uncertainty contribution of the random eects and run specic e

17、ects are assessed asmethod repeatability and run-to-run variation, respectively. The laboratory bias can beestimated from the method reproducibility obtained in large collaborative trails. In case ofsingle-laboratory validation a trueness test can be used to assess the combination of thelaboratory a

18、nd method bias 16. Two sources of measurement uncertainty that are specic for dPCR measurements arediscussed in greater detail in the following: the uncertainty contribution of the threshold settingand the uncer- tainty contribution of the assigned partition volume.The importance of the uncertainty

19、contribution of the threshold setting is determined by the number ofpartitions with intermediate uorescent amplitude (meaning between the uorescent amplitude of thenegative and the positive partitions). The causes of this intermediate uorescent amplitude are multiple,including abnormal sized partiti

20、ons, presence of PCR inhibitors, reduced accessibility of the targetsequence, non-specic amplication and incomplete mixing of PCR reagents. During the methoddevelopment, eorts should be made to reduce the amount of partitions with intermediate uorescentamplitude to a minimum by a good selection of t

21、he primer and probe sequences, careful titration of primerand probe concentrations and optimisation of the PCR conditions. Improving the quality of the analysedsamples might also have an eect. During the method validation, the amount of partitions with intermediateuorescent amplitude should be quant

22、ied both in samples with a high and a low copy numberconcentration of the target sequence to get information about their proportion compared to the positiveand negative partitions, respectively. Sup- plementary data Fig. 1 shows the results of a theoreticalsimulation for the ddPCR system in which th

23、e maximum uncertainty related to the threshold setting iscalculated for dierent proportions of rain droplets. In case of very low proportions of rain droplets (e.g.0.01 % of the negative droplets or 0.1 % of the positive droplets) the maximum uncertainty related to thethreshold setting can be consid

24、ered negligible compared to the other uncertainty contri- butions. However,for ddPCR methods with higher proportions of rain droplets the threshold-related uncertainty mightbecome a sig- nicant contributor to the overall measurement uncertainty. The uncertainty related to thethreshold setting is not

25、 constant over the whole working range of a dPCR method and will be larger at thelimits. The uncertainty of the partition volume contributes to the overall measurement uncertainty of a dPCRmeasurement when absolute copy number concentrations are measured. The manufacturer often provides the partitio

26、n volume of a dPCR system without any information about theassociated uncertainty. Results of inde- pendent attempts to verify the partition volume and to estimate the associated uncertaintyhave been described for dierent dPCR systems 22,29,31,32.The uncertainty on the partition volume is probably o

27、ne of the major reasons of the measurement biasbetween dierent dPCR systems as also observed during the method validation presented above.In the case of the ddPCR sys- tem, the volume of the droplets could be inuenced by the type of samples,the cartridges and the instruments, but especially by the t

28、ype of supermix, which plays a major role 32. Therefore it is important to use the droplet volume which is the most appropriate for the specic ddPCRmethod and to include the uncertainty associated with this droplet volume in the overall measurementuncertainty budget. 質(zhì)量評(píng)估Limitofdetectionandquanticat

29、ionThe measurement principle of the dPCR makes it possible to detect up to one copy of a target sequence and the Poisondistribution is applicable at these very low copy number concentrations. Therefore it is more reasonable to assess theLOD and LOQ by performing many replicate measurements at very l

30、ow copy number concentrations. The assessment should start by clearly dening the level of condence appropriate for the intended use of the method. Incase that the copy number concentration of the target sequence in the routine samples will be always well above theLOD, it is sucient to have a rough e

31、stimate of the LOD as done in the method validation presented above. When performing an experiment with 60 replicate measurements exactly at the LOD there would be on average 3 negativemeasurement results, as this represents 5 % of the cases. If all of the 60 replicate measurements performed at a ce

32、rtainPCR copy number concentration are positive, it can be reasonably assumed that this concentration is above the LOD ofthe method. More accurate estimates of the LOD are required for dPCR methods that are intended to be used to measure samples inwhich the target sequence could be absent, and this

33、absence would lead to rel- evant decisions. In this case, negativemeasurement results should be reported as copy number concentration LOD with a specied condence level.The LOD and LOQ of a dPCR method depend on the num- ber of analysed partitions and the total volume of the analysedpartitions. In ca

34、se of the ddPCR system the number of analysed partitions varies. The impact on the LOD and LOQ can beillustrated by calculating the theoretical minimum LOD based on the Poisson distribution, both at the level of sampling andthe distribution of the target sequence over the droplets. In the case of 15

35、 000 analysed droplets with a droplet volume of 0.834nL, the minimum theoretical LOD will be 0.32 cp/ Lin the PCR mix, as 5 % of the measurements at this level will not have a single copy of the target sequence in theanalysed droplets. In case that the number of ana- lysed droplets is 10 000, the mi

36、nimum theoretical LOD will increase to0.44 cp/ L. Careful manipulation of the droplets and rejection of measurement results with a low number of accepteddroplets (as done in this study by applying the technical reason exclusion crite- ria mentioned in Section 2.3) are thereforerequired to guarantee

37、a certain LOD and LOQ.The method validation described here can be used as an example for other singlelaboratory validations of dPCR methods. However, the extensiveness of a methodvalidation should always depend on the intended use of the method and on theacceptable level of measurement uncertainty.

38、The experiments should be conducted ina manner which provides a realistic view of all the factors pos- sibly aecting themeasurement result during routine use of the method, as well as covering theconcentration ranges and sample types within the scope of the method 24. The mostchallenging part of the

39、 validation of dPCR methods is probably the verication of thetrueness, as representative samples with a reference value are often dicult to nd. Thedevelopment of suitable certied ref- erence materials will diminish this problem andcan promote the transition of newly developed dPCR methods into relia

40、ble analyticalmethods suitable for diagnostic or other routine testing purposes.質(zhì)量評(píng)估Sample qualityTissue/切/blood/唾液DNA/RNA qualityQC mehtods常DNA質(zhì)控法對(duì)DNA評(píng)估參數(shù)法適DNA來(lái)源全部DNA可擴(kuò)增 DNADNA 純度Spectrophotometry冰凍新鮮組織、全、細(xì)胞系X冰凍新鮮組織、全、細(xì)胞系Fluorimetry(eg. Picogreen)XXFFPE組織、漿、清以及其他來(lái)源度降解DNAXqPCR31標(biāo)本因素影響外周漿臨床分檢測(cè)法質(zhì)量評(píng)估主要

41、指標(biāo)靈敏度/敏感度 Sensitivity疾病組 對(duì)照組分析靈敏度 Analytical sensitivity臨床敏感度 Clinical sensitivity真陽(yáng)假陽(yáng)測(cè)試陽(yáng)性測(cè)試陰性(a)(c)假陰真陰(b)(d)檢出/定量限度 LOD/LOQ: Limit of Detection/Quantitation （95%）特異性 Specicity分析特異性 Analytical specicity臨床特異性 Clinical specicity準(zhǔn)確度 Accuracy真實(shí)度 Trueness 精密度 Precision (5-10%)重復(fù)性 Repeatability 重現(xiàn)性 Repro

42、ducibility線性范圍 Linear dynamic range擴(kuò)增效率 PCR eciencySource: ISO 5725數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估靈敏度/敏感度 Sensitivity分析靈敏度 Analytical sensitivity臨床敏感度 Clinical sensitivity檢出/定量限度 LOD/LOQ: Limit of Detection/Quantitation （95%）多數(shù)情況：分析靈敏度 Analytical sensitivity = 檢出限度 LOD基因表達(dá)稀有序列稀有突變拷數(shù) 或濃度拷數(shù) 和突變例拷數(shù)Source：Biorad數(shù)字PCR檢測(cè)法質(zhì)量

43、評(píng)估線性范圍 Linear dynamic range（115490 copies per total reaction volume）Interexperiment linearity for qPCR(a) and the QX100 system(b)Anal Bioanal Chem. 2016 Jan;408(1):107-21.數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估LOD 和 LOQthe LOQ based on the interexperiment data was determined as thelowest nominal DNA concentration where the CV was still below 25 %Anal Bioanal Chem. 2016 Jan;408(1):107-21.數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估TruenessPLoS One. 2013; 8(5): e62583.數(shù)字PCR檢測(cè)法質(zhì)量評(píng)估PML-RARA quantication by ddPCRFigure 1 ddPCR-based detection of PML-RARA in serial dilutions（200ng cDNA input)J Mol Diagn. 2017 May;19(3):437-444