Laboratory medicine and in vitro diagnostics

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Details : C18RMP1 - NIM Reference measurement procedure for ''BRAF'' V600E (1799T>A) fractional abundance

Type (acronym)	rmp
Type	reference measurement procedure
Unique Nomination Number	C18RMP1
Analyte Name	Single nucleotide variant (BRAF V600E) fractional abundance
Method capable of Traceability of Analyte to SI or Defined Procedure	SI-traceable
Name	NIM Reference measurement procedure for BRAF V600E (1799T>A) fractional abundance
Organization that developed Validated Method	National institute of metrology, China
Measurement Techniques Used	Digital PCR
Comments 1	The reference measurement procedure gives BRAF V600E (1799T>A) fractional abundance of the total BRAF gene
Comments 2	We have participated an international comparison, CCQM P184 which is about the BRAF V600E measurement and satisfied result was obtained by using our proposed RMP
Quantity	Amount-of-substance ratio (DNA copy number ratio)
From (0)	0.1
To (0)	100
Unit (0)	%
From (1)	32
To (1)	1.8
Unit (1)	%
Level of Confidence (%)	95%
Peer Reviewed Publication	Interlaboratory assessment of droplet digital PCR for quantification of BRAF V600E mutation using a novel DNA reference material, Dong L. et al, Talanta 2020 207 120293
Comments to be published via JCTLM DB	This reference measurement procedure (RMP) specifies the operating specifications for measurement of fractional abundance (FA) of DNA containing BRAF single nucleotide variant (SNV) corresponding to amino acid mutation p.V600E by digital PCR, and is specifically designed for this SNV and its corresponding wild-type sequence: COSMIC Identifier: Genomic Mutation ID: COSV56056643 (Legacy Identifier COSM476) BRAF coding sequence: NM_004333.5: c.1799T>A Genomic coordinates (SNV): GRCh38, 7:140753358 Genomic coordinates (dPCR mplicon): GRCh38, 7: 140753312…140753382 The RMP is only suitable for DNA samples which have been confirmed by sequencing to containing the above SNV; it is not suitable for samples where the BRAF sequence variant is unknown. This method is applicable to the measurement of target BRAF p.V600E FA in plasmid and genomic DNA in buffered solution. The RMP has been tested with materials containing fragmented gDNA^[1],[2], however caution should be exercised when applying the RMP to fragmented DNA (<1 kb). The mutant and wild-type sequence-containing templates should have approximately the same fragment size distribution to avoid bias in the measurement of FA. DNA extracts from biological matrices should be checked for the presence of reaction inhibitors by checking purity by UV spectrophotometry and/or spiking methods^[3]. It is recommended that plasmid DNA is linearised and high MW gDNA is subject to restriction digestion prior to dPCR^[2]. The measurement interval of the RMP for FA is between 0.10% and 100%. The Limit of Blank (LoB), Limit of Detection (LoD) and Limit of quantification (LoQ) for FA were determined to be 0.010%, 0.020% and 0.10%, respectively. [1] Talanta 2020 207 120293 [2] Evaluation of droplet digital PCR and next generation sequencing for characterizing DNA reference material for KRAS mutation detection, Dong et al, Scientific Reports 2018 8 9650 [3] ISO 20395: 2019—Biotechnology—Requirements for evaluating the performance of quantification methods for nucleic acid target sequences— qPCR and dPCR
Analyte Category Name	Nucleic acids
CCQM Key Comparison Report	CCQM P184 Standardisation of cell-free DNA measurements: An international study on comparability of low concentration DNA measurements using cancer variants
Non-CCQM Key Comparison Report	See Talanta 2020 207 120293 for national interlaboratory study
Measurement principle/technique	Digital PCR