Amplification product leakage can lead to severe laboratory contamination. Follow these steps to eliminate contamination:
① Prioritize ventilation to ensure the smooth diffusion of amplification product fragments;
② Treat suspicious equipment by wiping or soaking with 1mol/L hydrochloric acid to depurinate residual DNA;
③ Use UV irradiation with a wavelength of 254/300 nm (Note: UV only works for long fragments over 500 bp with little effect on short ones);
④ If contamination persists for a long time, replace PCR reagents with products from another manufacturer for testing.

To judge whether the PCR instrument works normally, consider the following aspects:
1. Check if the instrument can be turned on/off normally and connected to software stably;
2. Verify if the runtime for the same experiment is consistent with usual to judge the normal operation of the heating and cooling module;
3. Check if the amplification curve shows a typical S-shape. An abnormal zigzag rising curve may indicate issues with the heated lid, fluorescence detection device or voltage;
4. If the fluorescence intensity is significantly lower than usual, consider replacing the instrument light source (especially halogen lamps with a lifespan of about 2000h);
5. If the fluorescence intensity of one or several wells is significantly higher than others, the well slots or detection optical path may be contaminated by fluorescence. Clean and calibrate the well slots before retesting.

1) A PCR laboratory should have sufficient and reasonable space, good lighting and air conditioning equipment. Although the working environment is not a direct factor of detection quality, a spacious and comfortable laboratory environment is certainly conducive to quality assurance;
2) Manage instruments and equipment reasonably and effectively with regular maintenance and calibration to keep them in good condition (e.g., calibrate pipettes regularly for sufficient accuracy and precision; maintain the optical system of real-time fluorescent PCR instrument and temperature difference between reaction wells within allowable ranges; also include the management of balances, centrifuges, refrigerators, etc.);
3) Select ideal reagents by controlling internal factors (sample processing methods, raw materials for nucleic acid amplification, methodological design, etc.) and external factors (kit transportation and storage conditions);
4) Establish standardized operating procedures: A complete PCR process includes sample collection, transportation, storage, numbering, reagent preparation, nucleic acid extraction, amplification, product detection, result analysis and reporting. Formulate scientific laboratory-specific SOP documents and implement them strictly;
5) Identify major pollution sources and take prevention measures: Main PCR pollution sources include a large number of target microorganisms in samples, cloned plasmids, specific microorganisms in the laboratory and residual contamination from previous amplification products (the main causes of false positives). Strictly partition the laboratory, follow the workflow, clean workbenches with chemical agents, and eliminate amplification product contamination by UV irradiation and UNG method;
6) Conduct internal and external quality control: Internal quality control ensures the consistency of in-lab testing results; external quality control provides data for retrospective comparison of laboratory testing results with objective inter-laboratory standards.

A standard real-time fluorescent PCR amplification curve includes baseline phase, exponential amplification phase, linear amplification phase and plateau phase, showing a typical S-shape. Analyze irregular curves according to specific conditions:
- Sloped rising curve: Malfunction or loose heated lid leading to inaccurate temperature control and liquid evaporation. Solution: Replace the heated lid or fasten it tightly;
- Discontinuous curve: Baseline end exceeds Ct value, usually caused by high template DNA concentration (CT<15) with baseline still set at 3-15 cycles (including partial amplification signals and suppressing the curve). Solution: Shorten the baseline end to 4 cycles before the Ct value and reanalyze the data;
- Linear amplification curve: Partial degradation of probes in some samples. Solution: Replace reagents for retesting;
- Plateau phase decline: Loose reaction tube cap. Solution: Check and fasten the PCR reaction tube cap tightly after covering.

5.1 False Negative Judgment
There are many causes for false negative results, mainly characterized by no FAM amplification curve, including sample inhibition, nucleic acid extraction failure, gene mutation and instrument malfunctions. Most mainstream domestic PCR reagents do not have internal control monitoring function, and the FAM amplification curve for target gene detection is used for both quantification and qualitative analysis (negative/positive judgment), so the quality of FAM amplification curve is critical.

For reagents with competitive internal control monitoring function, the following four scenarios may occur:
① No FAM amplification but normal internal control amplification: Normal result, judge the sample as negative;
② No FAM amplification and no internal control amplification: Abnormal result, possibly caused by nucleic acid extraction failure or inhibition, retest is mandatory;
③ Normal FAM amplification and normal internal control amplification: Normal result, as the target nucleic acid and internal control are amplified in the same reaction system;
④ Normal FAM amplification but no internal control amplification: Normal result, highly positive samples will inhibit internal control amplification, leading to weak or negative internal control results.

5.2 False Positive Judgment
False positive results are relatively rare in PCR detection, caused by reagent contamination, aerosol contamination, operational contamination, amplification product contamination, non-specific amplification, consumable contamination, etc. Therefore, it is recommended to set negative controls to monitor the presence of contamination.

(1) Popularity of use: 
For non-new brands, to ensure sufficient reliability of the purchased instrument, PCR laboratories can judge by the popularity of the instrument at home and abroad. More widely used instruments are more verified by practice and thus more reliable;
(2)Detection throughput (number of reaction wells): 
Select instruments with different throughputs according to laboratory requirements when purchasing quantitative PCR instruments. The detection throughput of real-time fluorescent PCR instruments on the market ranges from 16 to 384 wells. Laboratories can choose suitable instruments according to their testing projects and development status, and there is no need to pursue the most expensive ones. Generally, 96-well throughput is sufficient; adjust according to actual situations;
(3)Number of detection channels: 
With the increasing number of projects (e.g., gene expression, single nucleotide polymorphism analysis, high-resolution melting curve analysis), single-channel instruments cannot meet laboratory needs, while multi-channel design facilitates the detection of multiplex PCR and its derivative analysis modes such as gene expression;
(4)Consumable openness: 
The openness of consumables such as amplification reaction tubes may determine the level of daily testing costs. PCR laboratories can consider this when selecting real-time fluorescent PCR instruments. However, RNase-free consumables should be used for projects detecting RNA as pathogen genetic material;
(5) Hardware design features: 
Real-time fluorescent PCR instruments are mainly divided into 96-well plate type and centrifugal type, each with unique advantages and inevitable defects. 96-well plate instruments can hold a large sample volume (up to 100ul) without special consumables. Traditional 96-well plate instruments mostly adopt halogen lamp excitation and CCD detection, with advantages of higher light intensity and wider wavelength range than LED lamps (excellent for fluorescent dye excitation, stable performance, lifespan of about 3000 hours), but CCD detection may cause edge effects due to different optical paths from each sample well to the light source and detector, affecting results to a certain extent. Centrifugal instruments usually adopt LED excitation and PMT detection, avoiding edge effects in design. PMT detection can amplify or reduce fluorescent signals, being more flexible and sensitive than CCD detection, but LED has lower fluorescence intensity and narrower wavelength range, resulting in poorer excitation effect on fluorescent dyes than halogen lamps;
(6)Running speed: 
Among the many technical parameters of real-time fluorescent PCR instruments, the temperature rise and fall speed is also very important. Faster temperature rise and fall can shorten reaction time, reduce possible non-specific binding and reaction time, and improve PCR specificity. However, faster running speed has higher requirements for the heating and cooling module, so stability is the biggest problem. For instruments with long-term market verification and good stability, faster running speed brings more convenience;
(7)Flexibility: 
On the premise of guaranteed basic performance, consider the instrument flexibility, mainly including transportation flexibility, disassembly flexibility, software upgrade flexibility, module replacement flexibility and operation flexibility.