A PCR-lateral flow assay system based on gold magnetic nanoparticles for CYP2C19 genotyping and its clinical applications

Figures & data

Table 1. Primer sequences used for DNA sequencing, site-directed mutagenesis.

Allele	Application	Primer	Sequence
CYP2C19*2  (681G > A)	Sequencing	*2-seq-F	5′-CAACCAGAGCTTGGCATATTG-3′
		*2-seq-R	5′-CAATAAAGTCCCGAGGGTTGT-3′
CYP2C19*3  (636G > A)	Sequencing	*3-seq-F	5′-AGGCTGTAATTGTTAATTCGAGA-3′
		*3-seq-R	5′-TGTACTTCAGGGCTTGGTCA-3′
	Site-directed mutagenesis	Mp1 (F)	5′-AGGATTGTAAGCACCCCCTGAATCCAGGTAAGGCCAAGTTT-3′
		Mp2 (R)	5′-AAACTTGGCCTTACCTGGATTCAGGGGGTGCTTACAATCCT-3′

Table 2. Primer sequences used for ARMS-PCR.

Allele	Primer	Sequence	Mismatch	Mark
CYP2C19*2  (681G > A)	*2F	5′-ACAACCAGAGCTTGGCATATTGT-3′	–	5′-biotin
	*2R(M)	5′-GGTTTTTAAGTAATTTGTTATGGGTTCGT-3′	Penultimate	5′-Digoxin
	*2R(WT)	5′-TTTTTAAGTAATTTGTTATGGGTTCGC-3′	Penultimate	5′-Digoxin
	*2R(M1)	5′-GGTTTTTAAGTAATTTGTTATGGGTTGCT-3′	Antepenultimate	5′-Digoxin
	*2R(WT1)	5′-TTTTTAAGTAATTTGTTATGGGTTGCC-3′	Antepenultimate	5′-Digoxin
	*2R(M2)	5′-GGTTTTTAAGTAATTTGTTATGGGTTCAT-3′	Penultimate	5′-Digoxin
	*2R(WT2)	5′-TTTTTAAGTAATTTGTTATGGGTTCAC-3′	Penultimate	5′-Digoxin
CYP2C19*3  (636G > A)	*3F	5′-TGTGCTCCCTGCAATGTGAT-3′	–	5′-biotin
	*3R(M)	5′-AAAAAACTTGGCCTTACCTGGAAT-3′	Penultimate	5′-Digoxin
	*3R(WT)	5′-AAAAACTTGGCCTTACCTGGAAC-3′	Penultimate	5′-Digoxin
	*3R(M1)	5′-AAAAAACTTGGCCTTACCTGGTTT-3′	Antepenultimate	5′-Digoxin
	*3R(WT1)	5′-AAAAACTTGGCCTTACCTGGTTC-3′	Antepenultimate	5′-Digoxin
	*3R(M2)	5′-AAAAAACTTGGCCTTACCTGGAGT-3′	Penultimate	5′-Digoxin
	*3R(WT2)	5′-AAAAACTTGGCCTTACCTGGAGC-3′	Penultimate	5′-Digoxin

Figure 1. Schematic of the PCR-GoldMag LFA system. (a ) Structure of the lateral flow assay strips. (b) Principle of SNP genotyping. Each SNP (CYP2C19*2 or *3) detection using two complementary reactions (M tube and WT tube). (c) Principle of the PCR-GoldMag LFA System.

Figure 2. Accuracy of the PCR-GoldMag LFA system. (a) Six CYP2C19 genotypes were confirmed by DNA sequencing. (b) Six CYP2C19 genotypes were screened out by the PCR-GoldMag LFA system. (c) Based on the magnetic characteristics of GMNP, the results (negative, positive, and CYP2C19 *1/*1) were read by magnetic reader.

Figure 3. Specificity and sensitivity of the PCR-GoldMag LFA system. (a) The specificity results of LFA system presents. (b) Different DNA quantities of CYP2C19*2 and CYP2C19*3 heterozygous mutation genotype presents the sensitivity of LFA system. (c) Schematic of the sensitivity results of magnetic reader. (d) The magnetic signals of different DNA quantities of CYP2C19*2 and CYP2C19*3 heterozygous mutation genotype.

Table 3. The coincidence rate of PCR-GoldMag LFA system and DNA sequencing.

	Genotype	DNA sequencing						Total	Agreement (%)
		*1/*1	*1/*2	*1/*3	*2/*2	*2/*3	*3/*3
PCR-GoldMag LFA system	*1/*1	554	0	0	0	0	0	554	99.82
	*1/*2	1	494	0	0	0	0	495	100
	*1/*3	0	0	92	0	0	0	92	100
	*2/*2	0	0	0	127	0	0	127	100
	*2/*3	0	0	0	0	76	0	76	100
	*3/*3	0	0	0	0	0	12	12	100
	Total	555	494	92	127	76	12	1356	99.93

Figure 4. Pie chart for the genotype and allele frequency of CYP2C19 with 1356cases. (a) Gene frequency of CYP2C19. (b) Metabolic type of EM, IM, and PM. (c) The sample size data for genotyping of two provinces. (d) Genotype frequencies of CYP2C19*2 and CYP2C19*3.