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Quality control of Roche nucleic acid detection system

Date:2021/01/06 14:03:02
Viral nucleic acid detection technology (NAT) is a technology for direct detection of pathogen nucleic acid. With the comprehensive coverage of nucleic acid detection technology in blood stations, quality assurance and quality control have become the soul and core of nucleic acid detection. The factors that affect the quality of nucleic acid testing can be divided into three major factors: before, during, and after testing. These factors interact with factors such as personnel, equipment, materials, methods, and environment to affect the quality of nucleic acid testing. Therefore, single-factor control and quality analysis can no longer guarantee the quality of nucleic acid testing. A complete quality management system needs to be established to control the entire process. This can also effectively prevent viral diseases transmitted through blood transfusion and reduce the risk of virus transmission through blood transfusion.
With the comprehensive and deepening of blood nucleic acid testing in the national blood system, nucleic acid testing has strong specificity and high sensitivity, and the establishment and improvement of the quality system is particularly important [1]. The content of quality control covers all the steps of obtaining the inspection results, including the entire inspection process specimen collection, testing to the issuance of report results. Changsha Blood Center has carried out nucleic acid blood screening work since 2015. It has the Roche blood screening system of foreign diagnostic reagents and adopted a 6-sample mixed detection program. Now we summarize the key points that affect the quality of nucleic acid testing in daily work, and further standardize the inspection personnel The operation of the nucleic acid amplification detection system improves the detection quality and achieves the goal of continuous improvement.
1 Pre-measurement factors
1.1 Specimen collection
Train the collection staff for all staff. First save the enzyme tube, and then the nucleic acid tube. a low-temperature centrifuge to centrifuge. After leaving the sample, cover it upside down and mix for more than 8 times. The temperature of the sample before the test is cold chain transportation. 2~10℃, quality control should be done during the whole collection process.
1.2 Specimen reception
Receiving requirements for blood specimens: ①Specimen and blood correspond to each other; ②The quality of the specimens meets the technical requirements of the test items, carefully check whether the specimens have clots, not centrifuged, hemolysis, chyle, whether the test tube is damaged [2-4], whether the amount of specimens Sufficient amount and other phenomena, if the above situation occurs, carry out the relevant treatment measures and then carry out the experiment, the quality is unqualified, the type of anticoagulant, the order, quantity and method of the collection tube will affect the detection of nucleic acid in the blood sample; ③Specimen The information is traceable.
1.3 Laboratory facilities, instruments and equipment
The laboratory must do a good job in the maintenance and calibration of key instruments and equipment, including the nucleic acid extraction system, the amplification and interpretation system, and the maintenance and calibration of the micro sample gun. The repaired sampler, the light source of the thermal cycler, the temperature control system and other equipment should also be verified after failure, and the radiation intensity of the ultraviolet lamp should be monitored regularly. We need to pay special attention to the maintenance of the equipment during our daily experiments: ①75% ethanol to wipe the Core-Tip metal baffle and place it in the correct position; ②The barcode clip is only for one-time, and the barcode clip corresponds to the S-tube one by one. , The barcode holder needs to be put in place; ③Open the engineer panel of the CAP and observe whether there are large bubbles in the lotion connector 1 and lotion connector 2 or a section of the pipe joint is empty; ④ observe whether there are large bubbles and black particles at the top of the syringe Whether there are white crystals on the piston; ⑤ Whether there are droplets on the head of the reagent needle; ⑥ Preparation of the SPU (before on the machine, repeated confirmation after on the machine), the SPU must be placed in the correct direction, make sure that each SPU is close to the SPU rack on both sides, and upload Before the instrument, perform visual inspection on both sides of each SPU to avoid using brute force to slap the cover of the SPU (which will the S-tip to be stuck and cannot be pulled out and the machine to stop); ⑦K-tip and K-tube should be placed carefully, after placing Quickly confirm whether there is a consumable identification error on the Amplilink software. K-tip and K-tube racks can be placed in any position of the MP, K-tip and K-tube must be uploaded in full racks, and empty racks can be downloaded in CAP.
1.4 Ideal reagents and methods
Roche nucleic acid detection reagents are divided into detection reagents and control reagents. The detection reagents of the kit should be stored in the dedicated reagent storage area to prevent the reagents being contaminated. Before using the reagents, confirm the previous batch number, and pay attention to the difference in the coefficient of variation between the new and old batch numbers: Pay attention to the stability of the reagent: do not freeze the reagent, store MPX CS1, MPX CS2, MPX CS3 and MPX CS4 at 2~8℃; The valid period of opening the bottle is 20 d 2~8°C; cumulative time; 40 hours cumulative in COBASR AmpliPrep instrument, continuous time: continuous 24 hours in COBASR AmpliPrep instrument. "Standard operating procedures" should be written for reagent preparation, specimen collection, nucleic acid extraction, measurement methods and instrument operation.
1.5 Staff training
"Human" is the core of all work. Training activities for nucleic acid laboratory staff should be and implemented in strict accordance with standard operating procedures. Every staff member must have a nucleic acid laboratory "gene-free" or "nucleic acid-free" The concept, to prevent non-specific amplification results, the items and experimental records of each area are dedicated to the area, and must not be cross, understand the purpose and significance of each step of nucleic acid formulation, and strengthen the awareness of pollution prevention. If the experiment is invalid due to personal operation, it should be included in the personnel evaluation process, continuous improvement, and personal training should be increased. At the same time, it should increase the opportunities for personnel to go out for further training, learn cutting-edge and practical work experience to guide actual work, and continuously improve the staff’s Operation ability and data analysis ability.
2 Factors in the determination
2.1 Indoor quality control
Indoor quality control refers to a series of work taken by laboratory staff to determine the validity of the current batch of test results and whether the report can be issued. It is an immediate evaluation of laboratory measurements. Each analytical batch in the nucleic acid detection system must be tested once for quality control products. In nucleic acid extraction, a known weakly positive quality control sample should be taken. The test result is a comprehensive reflection of the effectiveness of nucleic acid extraction and amplification, negative The control has no amplification signal, the positive control amplification cycle number is within the normal range, and the internal standard amplification cycle signals of all samples are within the normal range. Dilute the quality control standards with concentrations of 200 IU/mL, 2 000 IU/mL, and 2 000 IU/mL to 2 to 5 times close to the lower limit of detection [5], mix well before the test, and monitor the experiment adding samples, nucleic acid extraction to amplification, it is best to indoor quality control products within 72 hours after thawing to ensure the stability of quality control products as much as possible. The amplification signal of quality control products is required to be within the expected range, and indoor quality control is established. Frame, observe the stability of quality control products, analyze the reasons for abnormal quality control values, common reasons for the out-of-control of positive quality control samples are: ① Random errors in nucleic acid extraction, such as incomplete removal of organic solvents, nucleic acid extraction Loss of the instrument, the consumables, such as PCR inhibitors in the centrifuge tube, residues of the amplification inhibitors in the specimen, etc.; ②Instrumental problems, such as the inhomogeneity of the temperature between the wells of the thermal cycler, and the inconsistency between the temperature in the well and the indicated temperature Reagent problems, such as the purity and labeling efficiency of probes, the efficiency of nucleic acid extraction reagents, the inactivation of Taq enzyme or reverse transcriptase, etc. [6].  2.2 Room quality evaluation
Inter-office quality evaluation is mainly to compare the results of laboratory quality evaluation, objectively evaluate the consistency and accuracy of the test results of the established items, understand the differences in test results, and propose targeted improvement measures. At present, the inter-laboratory quality assessment of the blood nucleic acid test of the center is mainly based on the relevant specimens provided by the Ministry of Health, the Provincial Clinical Examination Center, and the Australian inter-laboratory quality assessment. The test results are reviewed every time after participating in the inter-laboratory quality assessment. Timely feedback, compare the feedback results with the laboratory test results, and analyze the accuracy of the laboratory's nucleic acid test results.

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