Hepatitis C Virus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)_NMPA Approval in April 2018
Product Name: Hepatitis C Virus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)
Category: Domestic Class III
Registration Pathway: Innovation
NMPA Approval Time: April 2018
Product Structure and Composition: This kit includes nucleic acid extraction reagents, PCR amplification reagents, calibrators, quality control samples, and internal standards.
Scope of Application: This kit is used for the in vitro quantitative detection of hepatitis C virus (HCV) nucleic acid (RNA) in serum samples. It is suitable for patients who need to be tested for HCV infection and for hepatitis C patients receiving antiviral treatment.
The kit can detect common clinical genotypes of HCV, types 1 to 6. It is mainly used to assess the response and treatment effect of antiviral therapy by monitoring the content and changes of HCV RNA in the blood of hepatitis C patients. The detection should not be used as the sole indicator for evaluating the patient's condition, and a comprehensive evaluation of the patient's condition must be made in combination with clinical manifestations and other laboratory test indicators. This kit should not be used for HCV blood screening.
Packing Specification: 48 servings per box
Test Principle: In this kit, HCV RNA is extracted from serum samples using a lysis solution containing magnetic beads in the PCR amplification tube, and amplification is performed in the same tube. Under the action of reverse transcriptase, HCV RNA is reverse transcribed into HCV cDNA, and then under the action of DNA polymerase, the TaqMan probe technology is used to amplify HCV cDNA and perform real-time fluorescent quantitative detection.
The calibrators and quality control samples in this kit are clinical serum samples containing hepatitis C virus (inactivated).
This kit monitors whether there are PCR inhibitors in the sample by detecting internal standards to avoid false negatives.
Main Raw Materials:
1. The main raw materials of this product include primers, probes, UNG enzyme, dNTPs, etc., which are obtained through external purchase. M-MLV reverse transcriptase, Taq DNA polymerase, conformation magnetic beads, and RT-PCR buffer are self-prepared.
Primers and probes are designed by the applicant and synthesized by professional synthesis companies, and obtained after PAGE or HPLC purification; UNG enzyme is obtained after cloning, expression, and purification by raw material suppliers; dNTPs are obtained by direct chemical synthesis by raw material suppliers. M-MLV reverse transcriptase and Taq DNA polymerase are obtained after cloning, expression, and purification by the applicant, and conformation magnetic beads are prepared by the applicant. The RT-PCR buffer is prepared by the applicant.
2. The applicant selects the best raw materials and suppliers through functional tests from qualified suppliers. Quality requirements for each main raw material have been formulated and passed inspection. For self-prepared raw materials, the applicant has established quality standards and passed inspection.
3. Enterprise reference material setup
The applicant has designed a complete set of enterprise reference materials, including positive reference materials, negative reference materials, accuracy reference materials, detection limit reference materials, and precision reference materials. The reference materials are all prepared from clinical samples. The positive reference materials are derived from 10 different HCV RNA positive serum samples, covering different genotypes and interfering substances. The negative reference materials are derived from 10 different HCV RNA negative serum samples, covering samples from healthy populations, hepatitis B virus infection, HIV nucleic acid positive, HCMV/EBV, and other similar pathogen infections. The accuracy reference materials are prepared by gradient dilution of HCV RNA positive serum samples, which are L0 (1.0×10^6 IU/mL), L1 (1.0×10^5 IU/mL), L2 (1.0×10^4 IU/mL), L3 (1.0×10^3 IU/mL), and L4 (1.0×10^2 IU/mL), respectively. The detection limit reference materials are prepared by diluting HCV RNA positive serum samples with a concentration of 50 IU/mL. The precision reference materials are prepared by diluting HCV RNA positive serum samples, containing high and low concentration levels. The clinical samples used are all confirmed by marketed similar kits. Each enterprise reference material is used for product specificity, accuracy, sensitivity, and repeatability evaluation.
Research on Production Processes and Reaction Systems:
The applicant used the preliminarily determined formula to prepare the reaction system, and used enterprise reference materials/quality control samples to screen or optimize the HCV primer/probe concentration, internal standard primer/probe concentration, enzyme mixture usage, magnesium ion concentration, dNTPs concentration, RT-PCR buffer, reverse transcription time/temperature, annealing time/temperature, pre-denaturation time/temperature, denaturation time/temperature, sample usage, PCR applicable models (SLAN-96P, MX3000P/3005P and ABI7500 real-time fluorescence PCR instruments), nucleic acid lysis time, lysis solution usage, magnetic bead usage, magnetic bead adsorption time, washing solution concentration and washing times, selection of negative pressure pump flow rate, selection of magnetic rack magnetic materials and shelf life, etc., and finally determined the optimal production process and reaction system.
Analytical Performance Research:
The product's analytical performance includes nucleic acid extraction performance, limit of detection (LOD) and limit of quantification (LOQ), linear range, accuracy, precision, specificity (cross-reaction, interfering substances), etc.
The applicant submitted performance evaluation data for three batches of products on different applicable models.
In the nucleic acid extraction performance evaluation, the applicant compared the magnetic bead extraction method and pure nucleic acid spiking method for serum samples with high, medium, and low concentrations. The results met the performance requirements. HCV RNA high and low concentration serum samples were repeatedly tested 20 times each, and the coefficient of variation of the logarithmic values of the detected concentrations was ≤5%, meeting the repeatability requirements. HCV RNA strongly positive serum was diluted with HCV negative clinical serum containing high bilirubin, high lipids, hemolysis, and interferon to prepare high, medium, and low concentration samples, and compared with non-interfering serum samples. The results showed that the above interfering substances had no effect on the nucleic acid extraction performance.
In the LOD and LOQ performance evaluation, the applicant performed gradient dilution on HCV positive serum samples and conducted 20 repeated tests on a series of concentration samples. The lowest dilution concentration with ≥95% positive detection rate was used as the LOD, and the lowest dilution concentration with 100% detection rate and the difference between the logarithmic value of the concentration and the theoretical logarithmic value ≤±0.5 was used as the LOQ. The LOD of this product was finally determined to be 15 IU/mL, and the LOQ was 50 IU/mL. Repeated tests were performed on different HCV genotypes using three batches of reagents to verify that the kit's performance could meet the requirements of LOD of 15 IU/mL and LOQ of 50 IU/mL.
In the linear range performance evaluation, the applicant used clinical serum samples of different HCV genotypes to prepare 10 concentration gradient samples for linear range study. The concentration range of 50 IU/mL to 1.0×10^8 IU/mL showed a linear correlation, with a linear correlation coefficient > 0.980, meeting the performance requirements of the linear range.
In the accuracy performance evaluation, the applicant used HCV negative samples to serially dilute the national reference material and prepared five gradient samples of 1.0×10^6 IU/mL, 1.0×10^5 IU/mL, 1.0×10^4 IU/mL, 1.0×10^3 IU/mL, and 1.0×10^2 IU/mL for accuracy study. The results showed that the linear correlation coefficient was > 0.980, meeting the accuracy performance requirements.
In the precision performance evaluation, the applicant performed 20 repeated tests on precision samples and analyzed the results of intra-batch, inter-batch, inter-personnel, inter-instrument, inter-laboratory, and inter-day tests. The results showed that the coefficients of variation of the precision samples were all < 5%, meeting the precision performance requirements.
In the cross-reaction performance evaluation, the applicant evaluated the cross-reaction of human cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis A virus, syphilis, human herpesvirus 6, herpes simplex virus type 1, herpes simplex virus type 2, influenza A virus, Staphylococcus aureus, Candida albicans, West Nile virus, and dengue virus samples. The results showed that the above samples did not cross-react with this product.
In the interference experiment, the applicant used HCV RNA negative samples with total bilirubin ≥ 340 μmol/L, free hemoglobin ≥ 6 g/L, triglycerides ≥ 5 mmol/L, and interferon α-2a at 60 μg/L as diluents to prepare high, medium, and low concentration samples for repeated testing. The results did not interfere with the test results of this kit.
This application product only includes 1 packaging specification (48 tests/box), and the applicant provided performance evaluation data for three batches of products on all applicable models. The above performance studies meet the requirements of Announcement No. 44.
Research on Positive Judgment Value:
The applicant selected clinical serum samples covering genotypes 1 to 6 and used this reagent kit to study the positive cutoff value. They determined that the positive cutoff value is ≤40 Ct (cycle threshold).
Stability Research:
The applicant conducted research on the real-time stability, open-bottle and freeze-thaw cycle limit stability, transport stability, and sample stability of this product, determining the effective preservation time of the product and samples under various conditions. The reagent batches used included MC20150922, MC20150926, and MC20150929.
Real-time stability study: The nucleic acid amplification kit was stored at -20±5°C, and the nucleic acid extraction kit was stored at 2°C~8°C for 13 consecutive months. The appearance, positive and negative compliance rate, limit of detection, limit of quantification, accuracy, and precision of the kit were examined. All performance indicators met the requirements, determining that the product can be stably stored for 12 months under the specified storage conditions.
In addition, the applicant conducted separate studies on the open-bottle and freeze-thaw cycle limit stability, transport stability, and sample stability of the product. The results showed that the product's performance met the requirements stated in the product manual.
Clinical Evaluation:
The applicant completed clinical trials at three institutions: Capital Medical University affiliated Beijing You'an Hospital, Chinese People's Liberation Army 302 Hospital, and Capital Medical University affiliated Beijing Ditan Hospital.
A comparative study was conducted using the evaluated reagent and a marketed product to validate the clinical performance of this product on clinical samples. The enrolled samples included HCV RNA positive samples after clinical hepatitis C virus nucleic acid testing and HCV RNA negative samples from a non-hepatitis C diagnosed population, totaling 520 cases.
The comparative reagent selected was a similar marketed product (registration certificate number: National Food and Drug Administration (Import) No. 3401702 of 2014). For samples with inconsistent results between the evaluated reagent and the comparative reagent, a third-party reagent (registration certificate number: National Medical Device Registration No. 20153400085) was used for confirmation.
In this clinical trial, 520 serum samples were tested at three clinical trial units. Among them, two samples were from the same patient's serum, so one was excluded, and 519 samples were actually included in the statistics. There were 459 positive samples (88.4%) and 60 negative samples (11.6%). 43 samples had 15IU/mL to 50IU/mL. There were 5 samples of genotype 3a, 5 samples of genotype 3b, and 20 samples of genotype 6a.
Comparing the evaluated reagent with the comparative reagent, the positive agreement rate of the evaluated reagent was 99.1%, the negative agreement rate was 100.0%, and the total agreement rate was 99.2%. The positive agreement rate for samples with 15IU/mL to 50IU/mL was 93.0%.
Kappa test analysis showed a kappa value of κ=0.963, P<0.05, indicating that the evaluated reagent and the comparative reagent had very good consistency. Chi-square test showed P=0.125>0.05, indicating no statistical difference in the test results between the two detection methods.
Correlation analysis showed a linear correlation of r=0.994, P<0.05 between the evaluated reagent and the comparative reagent, indicating a good correlation between the two reagent kits. The linear regression equation obtained from regression analysis was Y=0.016+0.978X, with a 95% confidence interval for the slope: [0.968, 0.988]; and a 95% confidence interval for the intercept: [-0.037, 0.069]. Analysis of variance showed P<0.05, indicating that the linear regression between groups was statistically significant.
For samples with special genotypes (3a, 3b, 6a), the linear correlation coefficient between the evaluated reagent and the comparative reagent was r=0.997 (P<0.05), indicating a good correlation between the two reagent kits. The linear regression equation was Y = -0.106+0.999X, with a 95% confidence interval for the slope: [0.969, 1.029]; and a 95% confidence interval for the intercept: [-0.281, 0.068]. Analysis of variance showed P<0.05, indicating that the linear regression between the evaluated reagent and the comparative reagent was statistically significant.
In summary, the test results of the evaluated reagent and the comparative reagent were consistent, demonstrating equivalence.