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

False negative judgment: There are many reasons for false negative results, mainly reflected in the failure of FAM amplification curve, including sample inhibition, nucleic acid extraction failure, gene mutation and instrument failure. At present, domestic mainstream PCR reagents do not have the function of internal standard monitoring. The FAM amplification curve used to detect the target gene is used for both quantitative and qualitative purposes (to judge the yin and yang), so the quality of the FAM amplification curve is very critical. For such reagents without internal standard, it is recommended to combine the results of hepatitis B serum markers (five quantitative/qualitative results) to judge the yin and yang. Even if the FAM curve is amplified, this result should also be referred to, especially the result of E antigen. At the same time, for reagents with competitive internal standard monitoring function, the following four conditions can occur: if FAM is not amplified, it is marked with amplification: the result is normal, and the sample is judged to be negative; If FAM is not amplified and internal standard is not amplified: the result is abnormal, which may be caused by the failure of nucleic acid extraction or inhibition, the experiment must be repeated; If FAM is amplified, it is labeled with amplification: the result is normal, because the nucleic acid to be tested and the internal standard are amplified under the same reaction system; If FAM is amplified, but the internal standard is not amplified: the results are normal, and strong positive samples will inhibit the amplification of the internal standard, resulting in weak or negative internal standard results. False positive judgment (how to judge): There are few false positive results in clinical PCR testing, including reagent pollution, aerosol pollution, operation pollution, amplification product pollution, non-specific amplification, consumables pollution, etc., so it is recommended that a negative control must be made to monitor whether there is pollution.

In vitro diagnostic reagent products can be broadly divided into clinical chemistry, hematology, microbiology, immunology and other categories. Among them, clinical chemistry has the largest market share, nearly 40%; The second is immunology market, accounting for about 35%. Immunodiagnostic reagents are qualitative or quantitative diagnostic reagents based on the specific reaction of antigen and antibody. Such reagents have the fastest development in all diagnostic reagent products, no matter in technology or market. According to the diagnosis category, it can also be divided into infectious disease, endocrine, tumor, drug detection, immunology, blood type identification, etc. It is estimated that immunodiagnostic reagents will gradually replace clinical chemical reagents and become the mainstream of diagnostic reagents in the future. With the change of external factors, diagnostic reagents will not only become more diversified, but also develop towards household diagnostic reagents with strong specificity, high sensitivity, low price, simple use and diagnostic automation. Diagnostic reagents can be used in blood banks, hospitals, test centers, families, clinics and other departments. The product forms of diagnostic reagents are different due to different purposes. Diagnostic reagents are mainly used in hospitals and test centers, followed by families. The household diagnostic agent has the characteristics of simple, convenient and fast operation, and is suitable for those who have not received professional training. Diagnostic reagents such as pregnancy test, ovulation, blood sugar and urine used in general families belong to this kind of reagents. The proportion of such reagents in the overall market is not high, accounting for only about 9%. However, with the acceleration of people's pace of life, people pay more attention to efficiency, so such reagents are growing every year.        
Although diagnostic reagents currently account for only 25% of the biotechnology industry, they have a great impact on current medical diagnosis. As genetic engineering, gene recombination, single antibody and other biotechnology are constantly used to develop diagnostic reagents, in addition to increasing the sensitivity and specificity of reagents, it also makes it possible or rapid to diagnose infectious diseases, tumors or genetic abnormalities that were impossible or time-consuming in the past. In addition, the combination with automated analytical instruments or electronic technology not only makes these accurate diagnoses enter the clinical routine diagnosis stage from the research stage, but also shortens the distance between medical treatment and diagnosis.

Diagnostic reagents can be divided into two categories: in vivo diagnostic reagents and in vitro diagnostic reagents. It is usually a reagent for detection through the reaction between antigen and antibody. A: Classification of in vitro diagnostic reagents:
1、 In vitro biological diagnostic reagents managed by drugs include:
1. Blood type and tissue matching reagents;   
2. Microbial antigen, antibody and nucleic acid detection reagents;   
3. Tumor marker reagents;   
4. Immunohistochemical and human tissue cell reagents;   
5. Human gene detection reagents;   
6. Biochips;   
7. Allergic diagnostic reagents.   
2、 In vitro reagents managed by medical devices include:
1. Clinical basic laboratory reagents;   
2. Clinical chemical reagents;   
3. Reagents for blood gas and electrolyte determination;   
4. Vitamin testing reagents;   
5. Cytohistochemical staining agents;   
6. Autoimmune diagnostic reagents;   
7. Microbiological test reagents.
B: According to medical test items, clinical diagnostic reagents can be roughly divided into clinical chemical test reagents, immunological and serological test reagents, hematological and cytogenetic test reagents, microbiological test reagents, body fluid excreta and exfoliated cells test reagents, gene diagnostic test reagents, etc. Among them, clinical chemistry has the largest market share, nearly 34%; The second is immunology market, accounting for 29%. New immunodiagnostic reagents and genetic diagnostic reagents were developed in the late 1980s. They are the fastest growing diagnostic reagents in both technology and market.   
■ Clinical biochemical reagents
Clinical biochemical reagents mainly include several categories of products for the determination of enzymes, sugars, lipids, proteins and non protein nitrogen, inorganic elements, liver function, clinical chemical control serum, etc. They are mainly used to cooperate with manual, semi-automatic and general full-automatic biochemical analyzers and other instruments for detection, with single reagent, liquid dual reagent, dry powder dual reagent, chemical method reagent, standard products and other specifications. At the same time, all manufacturers provide testing room Quality control serum series for indoor quality control.   
■ Immunodiagnostic reagents
Immunodiagnostic reagents have the most varieties in diagnostic kits, and can be divided into infectious diseases, endocrine, tumor, drug detection, blood group identification, etc. according to the diagnostic category. From the perspective of results, it can be divided into EIA, colloidal gold, chemiluminescence, isotopes and other different types of reagents. Among them, radioimmunoassay reagents have been eliminated in the international market due to their environmental pollution, and a small amount of them are still used in China.     
■ Molecular diagnostic reagents
Molecular diagnostic reagents mainly include nucleic acid amplification technology (PCR) products that have been used in clinic and gene chip products that are currently being vigorously researched and developed at home and abroad. PCR products have high sensitivity, strong specificity, short diagnosis window, and can be used for qualitative and quantitative detection. They have been widely used for detection of hepatitis, sexually transmitted diseases, lung infectious diseases, eugenics, genetic disease genes, tumors, etc. However, due to market chaos and cross contamination, the Ministry of Health has strictly prohibited the clinical application of fluorescent electrophoresis qualitative PCR reagents, At present, the State Food and Drug Administration has approved a small number of hybrid semi quantitative and quantitative kits that have solved cross contamination. Gene chip is a combination of molecular biology, microelectronics, computers and other disciplines. It integrates a variety of modern high precision and cutting-edge technologies, and is praised by experts as the ultimate product in the diagnostic industry. However, it has high costs and is difficult to develop. At present, there are few types of products, which are only used for scientific research, drug screening and other purposes.

According to the health documents "Notice on the Batch and National Verification of In Vitro Immunodiagnostic Reagents for Blood Sample Testing of Blood Donors" (Weifa No. 10, 1994), "Notice on the Fixed Point Production of Anti A and Anti B Blood Grouping Reagents" (Weiyao No. 26, 1995), there are five kinds of blood source screening reagents prescribed by the national law: 1, A, B, O blood typing reagents; 2. Hepatitis B surface antigen ELISA reagent; 3. Hepatitis C virus antibody ELISA reagent; 4. HIV antibody ELISA reagent; 5. Syphilis diagnostic reagent.