SNT5326.4-2023 進(jìn)出口食品化妝品專業(yè)分析方法驗(yàn)證指南第4部分分子生物學(xué)方法

ICSCCSX中華人民共和國出入境檢驗(yàn)檢疫行業(yè)標(biāo)準(zhǔn)SN/T5326.4—進(jìn)出口食品化妝品專業(yè)分析方法驗(yàn)證指南4部分：分子生物學(xué)方法Guidelineonvalidationoftestmethodsforfoodandcosmeticsofimportandexport—Part4:Molecularbiologicalmethod 2024-07-01實(shí)中華人民共和國海關(guān)總 發(fā)SN/T5326.4— GB/T1.12020《標(biāo)準(zhǔn)化工作導(dǎo)1ISN/T5326.4—4T6379.2測量方法與結(jié)果的準(zhǔn)確度（正確度與精確度）2SN/T5326.11SN/T5326.220202SN/T5326.320203SN/T5326.1SN/T5326.22020根據(jù)所制定方法的預(yù)期用途選擇合適的驗(yàn)證參數(shù)，對于不同類型的分子生物學(xué)檢驗(yàn)方法，驗(yàn)1。1√√——√———√√√√√√√√a1SN/T5326.4—實(shí)驗(yàn)室內(nèi)驗(yàn)證由標(biāo)準(zhǔn)方法的起草單位完成，應(yīng)至少驗(yàn)證特異性、正確度和檢出限。實(shí)驗(yàn)室間實(shí)驗(yàn)室內(nèi)驗(yàn)證由標(biāo)準(zhǔn)方法的起草單位完成，需要驗(yàn)證方法的全部技術(shù)參數(shù)。實(shí)驗(yàn)室間驗(yàn)證由除標(biāo)準(zhǔn)起草單位以外的協(xié)同實(shí)驗(yàn)室完成，對于定量方法，至少需要驗(yàn)證方法的正確度、重復(fù)性和a）特異性：包容性檢測結(jié)果100%為陽性；排他性檢測結(jié)果100%為陰性；b）相對正確度≥95%。a）特異性：包容性檢測結(jié)果100%為陽性；排他性檢測結(jié)果100%為陰性。b）正確度：相對偏差2575回收率125%。c）重復(fù)性：相對標(biāo)準(zhǔn)偏差（RSDr）≤25%。d）再現(xiàn)性：相對標(biāo)準(zhǔn)偏差（RSDR）<35%。e）線性：R2系數(shù)0.98。f）擴(kuò)增效率：90110％SN/T5326.220205.1特異性包含包容性和排他性。包容性指能夠檢測出屬內(nèi)的幾個(gè)種或者種內(nèi)的幾個(gè)亞種，它體現(xiàn)了一個(gè)篩查方法的應(yīng)用范圍。排他性指方法能夠檢測出目標(biāo)靶基因，而不能檢測出所有可能發(fā)驗(yàn)證方法參照SN/T5326.3—20205.1.930至少需要20個(gè)已知在一般情況下對目標(biāo)基因有干擾的非目標(biāo)基因的樣品進(jìn)行排他性試驗(yàn)。驗(yàn)證樣品的濃度要求在LOD的100倍以上。定性方法的正確度是相對正確度（RT。相對正確度的驗(yàn)證至少需要60個(gè)樣品，其中陽性樣品數(shù)量大約占樣品總數(shù)25%~75%。檢測結(jié)果填入2。22

SN/T5326.4—T注：D+、DD示陽性參考樣品，D示陰性參考樣品；T+T，TP（T+/D+），F(xiàn)P（T+/D-），F(xiàn)N（T-/D+），TN相對正確度按公式（1）計(jì)算RT（TP+TN（TP+FP+TN+FN） SN/T5326.220205.2.5如果可獲得標(biāo)準(zhǔn)樣品，應(yīng)優(yōu)先采用標(biāo)準(zhǔn)樣品驗(yàn)證方法的正確度。對于每一種基質(zhì)，應(yīng)至少采3少進(jìn)行6如果無法獲得標(biāo)準(zhǔn)樣品，在空白樣品基質(zhì)中加入已知量的分析物，測定回收率。示例如下：a）選擇3181、210b）分析樣品并計(jì)算每個(gè)樣品中分析物的濃度，計(jì)算每個(gè)濃度水平的平均回收率。重復(fù)性驗(yàn)證要求采用正確度驗(yàn)證的濃度水平，每個(gè)濃度水平3~5個(gè)樣品，每個(gè)樣品重復(fù)測試2次~3次。每一次重復(fù)測試都是一次獨(dú)立的樣品測試（包括核酸提取和擴(kuò)增。13定量方法的重復(fù)性具體按照GB/T6379.25.2.3GB/T6379.2檢出限在方法測定范圍的低濃度范圍，按梯度倍比關(guān)系（80貝、60貝、40貝、201051拷貝）制備系列樣品，每個(gè)濃度1個(gè)樣品。每個(gè)樣品進(jìn)行10次測試。所有10次平行實(shí)驗(yàn)均為陽性時(shí)的一系列濃度中的最低濃度即為LOD3SN/T5326.4—定量限定量限驗(yàn)證試驗(yàn)與檢出限驗(yàn)證試驗(yàn)相同。LOQ是測試結(jié)果RSD低于25的一系列濃度中的最低濃度。RSD具體按照GB/T6379.2中的方法進(jìn)行計(jì)算。R2采用一個(gè)已知濃度的目標(biāo)靶基因樣品，進(jìn)行梯度倍比稀釋（340000340000貝、3400貝、1700貝、340貝、170貝、3472~3核酸擴(kuò)增試驗(yàn)，將稀釋后濃度的對數(shù)值與所得閾值循環(huán)數(shù)（Ct）值采用最小二乘法進(jìn)行線性擬合R2。擴(kuò)增效率是指一個(gè)循環(huán)后的產(chǎn)物增加量與這個(gè)循環(huán)的模板量的比值，其值位于0~1E=（10(-1/slope)- 式中：E1PCRMON88701DNA溶液進(jìn)行梯度稀釋，至DNA模板量分別為680000貝、68000貝、34000貝、68000貝、680貝、34貝、6834拷貝（34LOQ，測定結(jié)33DNA模板Ct123根據(jù)公式（2）計(jì)算得到3次重復(fù)試驗(yàn)的擴(kuò)增效率為，擴(kuò)增效率在90％~110％之間，4SN/T5326.4—ThisdocumentiswrittenaccordingtotherulesfroGB/1.1-2020StandardizationGuidelinesPart1:TheStructureandWritingRulesofStandards.ThisdocumentisthePart4ofstandard"SN/T5326Validationguidelinesofanalyticalmethodforimportedandexportedfoodandcosmetics",whichhadbeenissuedthefollowingparts:——Part1:Generalguideline——Part2:Chemicalmethods——Part3:Traditionalmicrobiologymethods——Part4:Molecularbiologicalmethod——Part5:ImmunologicalPleasenotethatsomeofthecontentsofthisdocumentmayinvolvepatents.Thepublisherdoesnottakeresponsibilitytoidentifythesepatents.ThisdocumentisproposedandmanagedbyGeneralAdministrationofCustoms,People’sRepublicofThisdocumentwasdraftedbythefollowinginstitutions:TianjinCustoms,TianjinNormaUniversity,DalianMinzuUniversity,ShaoguanCollege,ChinaPeople'sPoliceUniversityandShenzhenCustoms.Thisdocumentwasmainlydraftedbythefollowingpeople:LiangjuanZhao,WenjieZheng,QiuyueZheng,WeiLiu,HongweiZhang,JijuanCao,XiaZhang,HonghaiWang,ChaoJiandZhenfengYue.5SN/T5326.4—GuidelineonvalidationoftestmethodsforfoodandcosmeticsofThisdocumentprovidesthevalidationguidelinesformolecularbiologicalmethodsfornucleicacidamplificationtestofimportedandexportedfoodandcosmetics.Thisdocumentisapplicabletotheverificationofmethodsduringtheestablishmentofmolecularbiologicalmethodstandardsfornucleicacidamplificationtestofimportedandexportedfoodandcosmetics,whetherthestandardsarenewlyformulated,revised,orequivalentormodifiedinternationalstandards.Inaddition,whenthelaboratoryadoptsnon-standardmethodsandindependentresearchanddevelopmentofnewmethods,itcanalsorefertothisguidetovalidatetheapplicabilityofthesemethods.NormativeThefollowingdocuments,inwholeorinpart,arenormativelyreferencedinthisdocumentandareindispensableforitsapplication.Fordatedreferences,onlytheeditioncitedapplies.Forundatedreferences,thelatesteditionofthereferenceddocument(includingallamendments)applies.GB/T6379.2,Accuracyofmeasurementmethodsandresults(truenessandprecision),Part2:BasicmethodsfordeterminingtherepeatabilityandreproducibilityofstandardmeasurementmethodsSN/T5326.1,Validationguidelinesofanalyticalmethodsforimportedandexportedfoodandcosmetic,Part1:GeneralguidelineSN/T5326.2-2020,Validationguidelinesofanalyticalmethodsforimportedandexportedfoodandcosmetic,Part2:ChemicalmethodSN/T5326.3-2020,Validationguidelinesofanalyticalmethodsforimportedandexportedfoodandcosmetic,Part3:TraditionalmicrobiologicalmethodTerminologyandThetermsanddefinitionsestablishedinSN/T5326.1andSN/T5326.2-2020areapplicabletothisPerformanceparametersofmolecularbiologicalSelectionofvalidationSelectionofvalidationSelectappropriatevalidationparametersaccordingtotheintendeduseoftheformulatedmethod.Fordifferenttypesofmolecularbiologicalmethods,theselectionofvalidationparametersisshowninTable1.7SN/T5326.4—Table1—SelectionoftypicalvalidationLimitofLimitof√√——√———√√√√√√√√athetruenessofthequalitativemethodisrelativeIn-housevalidationshallbecompletedbythedraftingunitofthestandardmethod,andthreeparametersincludingthespecificity,truenessandlimitofdetectionshallbevalidatedatleast.Theinter-labvalidationshallbecompletedbythecooperativelaboratory,otherthanthedraftingunitofthestandardmethod.Forqualitativemethods,atleastthetruenessofthemethodsshallbevalidated.QuantitativeIn-housevalidationshallbecompletedbythedraftingunitofthestandardmethod,andalltechnicalparametersofthemethodshallbevalidated.Theinter-labvalidationshallbecompletedbythecooperativelaboratory,otherthanthedraftingunitofthestandardmethodForquantitativemethods,aleastthreeparametersincludingthetrueness,repeatabilityandreproducibilityofthemethodsshallbevalidated.PerformanceThequalitativemethodshallmeetthefollowingperformanceSpecificity:100%oftheinclusivetestresultsarepositive;100%oftheexclusivetestresultsareRelativetrueness≥ThequantitativemethodshallmeetthefollowingperformanceSpecificity:100%oftheinclusivetestresultsarepositive;100%oftheexclusivetestresultsareTrueness:relativedeviation≤25%or75%≤rateofrecovery≤Repeatability:relativestandarddeviation(RSDr)≤Reproducibility:relativestandarddeviation(RSDR)<Linearity:R2coefficient≥Amplificationefficiency:90%≤amplificationefficiency≤ValidationofperformanceparametersofmolecularbiologicalThepreparationofvalidationsamplesshallrefertoSN/T5326.2—2020Chemicalmethods8SN/T5326.4—Specificityincludesinclusivenessandexclusivity.Inclusivenessreferstotheabilitytodetectseveralspeciesorsubspecieswithinagenus,whichreflectstheapplicationscopeofascreeningmethod.Exclusivitymeansthatthemethodcandetectthetargetgene,butcannotdetectallothergenesthatmayoccurcrossreaction.Thespeciesthatarefarfromthegeneticrelationshipwiththetargetgenespeciescanbeselectedtoevaluatetheexclusivity.ThevalidationmethodshallrefertoSN/T5326.3—2020Traditionalmicrobiologymethods5.1.9.Atleast30targetgenesamplesarerequiredforinclusivetest;Atleast20samplesofnon-targetgenesknowntointerferewiththetargetgeneingeneralarerequiredforexclusivitytest.Theconcentrationofthesampleusedforvalidationshallbemorethan100timesofLOD.TruenessofqualitativeThetruenessofqualitativemethodisrelativetrueness(RT).ThevalidationofRTrequiresatleast60samples,inwhich,thenumberofpositivesamplesaccountsforabout25%-75%ofthetotalnumberofthesamples.ThetestresultsshallbefilledinTable2.TruestateofPositivereferencesampleNegativereferencesampleDetectionNegativeresultD+),FPrepresentsfalsepositive(T+/D-),FNrepresentsfalsenegative(T-/D+),TNrepresentstruenegative(T-/D-).TherelativetruenessiscalculatedaccordingtoformulaRT（TP+TN（TP+FP+TN+FN） TrunenessofquantitativeValidationThevalidationmethodshallrefertoSN/T5326.2—2020ChemicalmethodsThestandardsampleshallbepreferredtoverifythetruenessofthemethoifthestandardsampleisavailable.Foreachmatrix,atleast3concentrationlevelsshallbeadopted,includingthelowconcentrationlevelandhighconcentrationlevelofthedeterminationrangeofthemethod;Foreachconcentrationlevel,atleast6parallelindependenttestsshallbeperformedtocalculatetherelativedeviationoftheaveragevaluefromtheacceptedreferencevalueofthestandardsample.9SN/T5326.4—TruenessofvalidationmethodswithadditionofstandardintotheIfthestandardsamplecannotbeobtained,addaknownamountofanalytetotheblanksamplematritodeterminetherateofrecovery.TheexamplesareasThreegroupsof18blankmatricesareselected,andtheanalyteswithabout1time,2timesand10timesthelimitofquantitationareaddedtoeachgroup.Analyzethesamplesandcalculatetheconcentrationofanalytesineachsample,andcalculatetheaveragerecoveryrateateachconcentrationlevel.Therepeatabilityvalidationrequiresthattheconcentrationleveloftruenessvalidationshallbeadopted.Eachconcentrationlevelincluding3samples~5samples,andeachsampleshallbetestedfor2time~3times.Eachrepeattestisanindependentsampletest(includingnucleicacidextractionandamplification).Note:3parallelamplificationfrom1nucleicacidextractisnotallowed.TherepeatabilityofquantitativemethodshallbecalculatedaccordingtothemethodinGB/TThereproducibilityvalidationofquantitativemethodshallbecarriedoutaccordingtostep5.2.3.ThereproducibilityshallbecalculatedaccordingtothemethodinGB/T6379.2.LimitofdetectionInthelowconcentrationrangeofthedeterminationrange,prepareaseriesofsamplesaccordingtothegradientratiorelationship(e.g.80copies,60copies,40copies,20copies,10copies,5copiesand1copy),with1sampleforeachconcentration.Eachsampleistested10times.Whenall10parallelexperimentsarepositive,thelowestconcentrationinaseriesofconcentrationsisLOD.LimitofquantificationThequantitativelimitvalidationtestisthesameasthedetectionlimitvalidationtest.LOQisthelowestconcentrationinaseriesofconcentrationswithRSDlessthan25%.RSDshallbecalculatedaccordingtothemethodinGB/T6379.2.LinearR2Atargetgenesamplewithknownconcentrationisadoptedtoconductgradientdilution(e.g.340000copies,34000copies,17000copies,3400copies,1700copies,340copies,170copies,34copiesand17copies).Thenucleicacidamplificationtestshallberepeatedatleast2times