Levey Jerming quality control chart is adopted, with X ± 2SD warning line and X ± 3SD out of control line. When the quality control point falls outside of x ± 3S, the first measure to be taken is re inspection. The purpose of re inspection is mainly to find out the human error, but also to find out the accidental error. If it is an accidental error, the re measurement results should be within the allowable range. At the same time, we also observed the generation of standard curve, amplification efficiency, curve ladder, curve shape, and Ct value drift, and combined with the detection results of clinical samples, whether there are high and low values, to distinguish between the loss of control caused by reagents or the loss of control caused by improper operation, instrument failure, or aging. If the curve standard of the clinical sample test results and the measured value are high and low, and the reagent blank and negative quality control are under control at the same time, it is likely that the quality of the standard product is defective, such as degradation, change of reagent model, etc. For seven consecutive quality control points falling on the side of the mean value, if none of them exceeds the range of X ± 3s, it is considered that there is a systematic error, but the test results can still be issued; If seven quality control points fall on one side of the mean value and the quality control data tends to exceed the range of X ± 3s (gradually increasing or decreasing), the instrument engineer must be contacted to calibrate the instrument in time.

Because the traditional quantitative methods are endpoint detection, that is, PCR is detected after reaching the plateau. However, when PCR reaches the plateau after logarithmic amplification, the detection reproducibility is very poor. The same template was repeated 96 times on the 96 well PCR instrument, and the results were very different, so the starting template quantity could not be calculated directly from the end product. After adding the internal standard, the inaccuracy caused by the final product quantification can be partially eliminated. But even so, the traditional quantitative methods can only be regarded as semi quantitative and rough quantitative methods. Internal standard quantification: If an internal standard with a known initial copy number is added to the sample to be tested, the PCR reaction becomes a dual PCR, and there is interference and competition between the two templates, especially when the initial copy number of the two templates is relatively different, the competition will be more significant. However, since the initial copy number of the sample to be tested is unknown, it is impossible to add an appropriate number of known templates as internal standards, which is precisely why the traditional quantitative method is still a semi quantitative method despite the addition of internal standards. External standard quantification: the external standard method is not to add the reference material to the tested sample, but to detect it under the same conditions as the tested sample. Because there is a linear relationship between the Ct value and the logarithm of the starting template in fluorescent quantitative PCR, the standard curve can be used to quantitatively measure unknown samples. When the PCR cycle just enters the real exponential amplification period, the reproducibility of the Ct value is excellent, that is, the same template is amplified at different times or in different tubes at the same time, the Ct value obtained is constant, so the quantitative results will be much more accurate. The scientific researchers of Texas University in the United States made a methodological comparison between the external standard method and the internal standard method, and concluded that the internal standard method is not reliable as a quantitative or semi quantitative method, while the external standard curve quantitative method is an accurate and reliable scientific method. 【Ke LD, Chen Z, Yung WK.A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards. Mol.Cell Probes,2000,14(2):127-135】

If the amplification products of the laboratory leak, causing the pollution of the laboratory, the consequences will be very serious. To remove the pollution, the first and most important thing is to maintain ventilation to ensure the smooth diffusion of amplified product fragments; Secondly, dilute acid treatment can be used to wipe or soak the suspected apparatus with 1mol/L hydrochloric acid to depurine the residual DNA; The UV irradiation method is adopted again. The UV wavelength (nm) is generally 254/300nm. It should be noted that when UV is selected to eliminate the residual PCR product pollution, the length of the PCR product and the distribution of bases in the product sequence should be considered. UV irradiation is only effective for long segments above 500bp, but not for short segments; Finally, if the contamination cannot be eliminated for a long time, it is recommended to replace the PCR reagent of another manufacturer for detection, because the primer design area of each manufacturer is different; Generally, the amplification products of one reagent company will not be in the primer design area of another manufacturer, so it will not cause false positive of product pollution.

1) In order to ensure the testing quality, the clinical PCR laboratory should have sufficient and reasonable space, good lighting and air conditioning equipment. Although the working environment is not the direct cause of testing quality, the spacious and comfortable laboratory environment will certainly be conducive to the assurance of testing quality.
2) The instruments and equipment shall be managed reasonably and effectively, maintained and calibrated regularly to keep them in good condition. For example, calibrate the pipette regularly to maintain adequate accuracy and precision. Regularly maintain the optical system of the fluorescent quantitative PCR instrument and the temperature difference between reaction holes to keep them in good condition and within the allowable range. In addition, it also includes the management of balance, centrifuge, refrigerator and other equipment.
3) Ideal reagent: There are two main factors, internal and external. The internal factors include sample processing methods, raw materials for nucleic acid amplification and methodological design. Outgoing factors include problems in the transportation and storage of the kit.
4) Standardized operation process: The complete clinical PCR procedure consists of sample collection, transportation, preservation, numbering, reagent preparation, nucleic acid extraction, amplification and product detection, result analysis and report, etc. The SOP document that is scientific and matched with the laboratory is established, and the operation is carried out in strict accordance with the SOP.
5) Main pollution sources and pollution prevention measures: The main pollution sources of PCR include a large number of microorganisms to be tested in the samples, cloned plasmids, a large number of specific microorganisms in the laboratory and residual pollution of previously amplified products. These are the most likely to cause false positive contamination in the laboratory. Therefore, it is necessary to strictly partition the laboratory and follow the workflow, use chemical cleaning of the experimental table, and use ultraviolet radiation and UNG methods to eliminate amplification product pollution.
6) Carry out indoor and inter room quality control. The indoor quality control can ensure the consistency of laboratory indoor measurement quality, and the inter room quality control can provide data for retrospective comparison between laboratory measurement and objective standards.

HBV-DNA test is the most direct indicator to reflect the degree of replication and infectivity of hepatitis B virus. Clinical data show that the detection rate of HBV DNA positive is very high in two pairs and a half of hepatitis B cases with three positive cases, but the result of HBV DNA negative is also very possible. Generally, the negative of hepatitis B big three positive HBV-DNA indicates that the patient's virus does not exceed the standard, but the big three positive result indicates that the patient is infected with hepatitis B virus and has strong infectivity. However, clinical examiners may also encounter the situation that HBV DNA cannot be detected in big three positive samples. First of all, such samples can be diluted and rechecked to exclude the presence of inhibitors in serum or plasma samples or the failure of PCR amplification caused by the concentration of HBV DNA in positive samples exceeding the upper limit of the kit. Secondly, the mutation of hepatitis B virus is also one of the reasons why the PCR amplification can not be achieved. Usually, the mutation of hepatitis B virus nucleic acid sequence occurs in patients treated with drugs. If multiple PCR reagents fail to detect, DNA sequencing can be carried out to determine hepatitis B virus and its mutation sites, so as to guide clinicians in drug treatment. Finally, the reagent of another manufacturer can be replaced for testing to eliminate the missed test caused by the design defect of the original reagent itself.

Real time fluorescent quantitative PCR amplification curve includes baseline period, exponential amplification period, linear amplification period and amplification platform period. The standard curve should be a typical S type. The irregular curve can be analyzed according to the specific conditions of the curve: 1) The reason why the curve rises in a diagonal line: the hot cover fails or is not tightly covered, resulting in inaccurate temperature control and liquid volatilization. Solution: Replace the instrument heat cover or tighten the heat cover. Fig. 1 The curve rises in a diagonal line. 2) The amplification curve is divided into two segments (breaks). Fig. 2 The amplification curve is divided into two segments (breaks). The reason is that the end point of the baseline is greater than the Ct value, usually because the template DNA concentration is high, the CT value is<15, but the baseline is still 3-15, which contains some amplification signals, causing the curve to be pressed down. Solution: Reduce the baseline endpoint to the first 4 cycles of Ct value, and re analyze the data. 3) Fig. 3 Linear Amplification Curve Cause of linear amplification curve in some samples: partial degradation of probe Solution: It is recommended to replace reagent for testing. 4) Reason for falling off during the curve plateau period: The cover is not tightly closed Solution: After the PCR reaction tube is closed, check whether the cover is tightly closed. Figure 4 Falling of Curved Platform Period