Analytical validation of GMEX rapid point-of-care CYP2C19 genotyping system for the CHANCE-2 trial

Study design and ethics approval

We evaluated the performance of the GMEX point-of-care testing system against conventional clinical laboratory-based genomic testing for CYP2C19 genotype detection. We conducted a multicentre study to assess the accuracy and result TAT between the two genotyping methods. Sanger sequencing is a gold standard test and was used to validate the genotyping results from two methods. The Ethics Committee of Beijing Tiantan Hospital Capital Medical University approved this study. All participants provided written informed consent.

Study subjects

Healthy individuals and patients with a clinical history of cardiovascular and cerebrovascular diseases were enrolled at six hospitals (Beijing Tiantan Hospital; Kaifeng Central Hospital; the Third People’s Hospital of Tongzhong District, Nantong City; the People’s Hospital of Wendeng District, Weihai City; Liaocheng people’s Hospital and Yixing people’s Hospital) between July and August in 2019. Inclusion criteria were healthy individuals by physical examination or patients with the ischaemic attack or other cardiovascular and cerebrovascular diseases. Individuals younger than 18 years of age and pregnant women were excluded.

GMEX point-of-care genotyping system

The GMEX point-of-care genotyping system was jointly developed by Chongqing Jingyin Bioscience and the China National Clinical Research Center for Neurological Diseases and was eventually transformed, produced and marketed by Chongqing Jingyin Bioscience. The GMEX system includes a portable DNA analyzer, genotyping reagents and a buccal sample collection kit. The system is user-friendly and can be easily operated, transmitted and analysed and presents an effective and simple approach to genotyping.

The operators associated with the GMEX system were doctors, nurses or clinical researchers. The GMEX system can be operated in the clinical department and bedside. Professional instructors trained all operators on-site and operators were assessed. Those that passed assessments obtained certificates. The training time for each hospital was about 2–4 hours.

The system uses noninvasive sampling by buccal swab at the bedside to improve patient compliance. It integrates automated steps of PCR-based amplification, fluorescent signal detection and genotype determination and is performed at the site of patient care by trained doctors, nurses or clinical researchers, dramatically improving the speed and user-friendliness of genotyping. The system has integrated controls to monitor the performance of a run and ensure ongoing quality of results. The innovative point-of-care test technology is in line with the affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users criteria proposed by the WHO.23 It is user-friendly, rapid and robust, equipment-free and deliverable to those who need them.

Sample collection and processing

Three buccal swabs and 2 mL peripheral blood in EDTA were collected from healthy individuals and patients. The buccal swabs were either analysed immediately or stored under the manufacturer’s recommended conditions prior to analysis by the GMEX system. Peripheral blood was subjected to analysis using the Human CYP2C19 Gene Polymorphism Detection Kit (YZYMED, Wuhan, China, referred as YZY Kit), a clinically proven method approved by the National Medical Products Administration (NMPA), at the central laboratory of Beijing Tiantan Hospital Capital Medical University and Sanger sequencing at Beijing Genomics Institute (BGI, Beijing, China) for validation. Gender, age and process time were recorded in the data collection form for each subject . Process time of laboratory-based genotyping was only available for subjects whose blood sample was collected at the Beijing Tiantan Hospital, because the blood sample would need to be transported from other centres to the central laboratory before testing.

Genotyping by the GMEX system

Buccal swabs were collected and analysed in accordance with the manufacturer’s instructions. In brief, buccal swab samples taken from the healthy individuals or patients were directly inserted into separately packaged reaction tubes including 23 µL of reaction mixture. This allowed simultaneous mixing of the samples with reagents for detecting CYP2C19 *2, *3 or *17 alleles and sealing of the reaction tubes. The samples were then subjected to analysis by the GMEX DNA analyser using the Taqman assay (Applied Biosystems, Foster City, California, USA). Primers and probes used for three single nucleotide polymorphisms are listed in table 1.

The GMEX DNA analyser is a portable fluorescent PCR machine with 12 reaction wells. Wells cannot be run independently and the temperature increase or decrease and fluorescence excitation or collection are performed at the same time for 12 reaction wells. The GMEX DNA analyser should not be paused while it is running and samples or reagents cannot be added midway.

The PCR conditions used were 95°C for 5 min, followed by 50 cycles of 95°C for 8 s and 55°C for 35 s. The genotyping results were interpreted with the accompanying software. The GMEX system was operated by nonlaboratory trained healthcare personnel.

Clinical laboratory-based genotyping

Genomic DNA from peripheral blood was extracted using the MagNA Pure 96 System (Roche, Basel, Switzerland) and subsequently analysed using the YZY Kit according to the manufacturer’s instructions. In brief, 2 µL (10–30 ng) of genomic DNA was added to a 23 µL reaction tube and subjected to quantitative PCR analysis using the LightCycler 480 PCR system (Roche, Switzerland). According to the criteria set by the YZY Kit manufacturer, the genotype was determined by professional laboratory personnel.

Validation of CYP2C19 genotypes

The genotypes of study participants were validated at the BGI Laboratory using Sanger sequencing. PCR primers (*2 forward: 5′-CAGAGCTTGGCATATTGTATC-3′ and *2 reverse: 5′-GTAAACACAAAACTAGTCAATG-3′, *3 forward: 5′-TGTGCTCCCTGCAATGTGAT-3′ and *3 reverse: 5′-TTTGGGGCTGTCACCAAAGT-3′,*17 forward: 5′-GCCCTTAGCACCAAATTCTC-3′ and *17 reverse: 5′-ATTTAACCCCCTAAAAAAACACG-3′) were designed to amplify CYP2C19*2, *3, and *17 genomic fragments. The PCR conditions were 96°C for 5 min, followed by 10 cycles of 96°C for 20 s, 62°C–52°C touchdown for 20 s and 72°C for 30 s and 35 cycles of 96°C for 20 s, 55°C for 20 s and 72°C for 30s. The final extension was performed at 72°C for 5 min. Peripheral blood samples were processed and subsequently analysed using a 3730XL DNA sequencer (Applied Biosystems, Massachusetts, USA).

Statistical analysis

Patient characteristics were described as medians with IQRs for continuous variables and frequencies and percentages for categorical variables. Kappa statistics were used for the assessment of diagnostic value agreement among the three platforms. The genotype was tested by Hardy-Weinberg equilibrium. All analyses were performed with SAS software V.9.4 (SAS Institute, Cary, North Carolina, USA).