Summary on PCR False Negative Problems

The problem of false positives in PCR is highly valued, but the problem of false negatives in PCR is more serious than clinical testing. As far as the problem of PCR false positives is concerned, generally only contamination and non-specificity of primers are involved. Pollution can be basically solved through strict laboratory management, reasonable environment settings, and adding UNG enzyme anti-pollution, while the non-specificity of primers basically belongs to the quality of the manufacturer's reagents. However, the false negative of PCR is different. It involves almost all personnel and technical aspects of PCR experiment, which is very complicated. From a clinical point of view, the detection rate of the three positives is low, and even the PCR test is still negative, which shows the seriousness of the false negative problem.

Generally speaking, there are the following factors on the problem of PCR false negatives:

1. Instrument factors

PCR experiments are highly dependent on instruments, and centrifuges and amplification instruments are all factors that cause false negatives in PCR. The main problem of the amplification instrument is the difference between the wells, which causes the failure of the amplification and the reduction of the amplification efficiency. The centrifuge effect is more easily overlooked. Domestic centrifuges rarely use centrifugal acceleration (XXXg) as a parameter index, but often use the number of revolutions as a parameter index. There is a problem here. Because the size of the centrifuge is different, the effective heartbeat radius is also different, so the same The centrifugal force generated by the rotating speed varies greatly (some are more than several times), so under the same centrifugation time, the template may not be centrifuged down, resulting in false negative PCR. This problem also exists in other experiments, but because the other experiments are to measure the precipitation of macromolecular proteins, the requirement for centrifugation speed is relatively low, so it is not obvious. It is recommended to pay attention to this problem in experiments using small desktop centrifuges.

2. Reagent quality problems

The quality of reagents is crucial to the success of PCR experiments. The quality of reagents involves many aspects: cell lysis; template extraction; primer site selection; Taq enzyme activity and so on. Any problem in any link will cause false negative results. These are all problems of reagent quality, so it is very important to choose high-quality PCR reagents.

3. Nucleic acid template problem

The quality of nucleic acid template is one of the important factors restricting PCR. Template quality problems such as fragmentation of the nucleic acid template in the amplification region, protein adhesion, and steric hindrance may cause false negative results or inaccurate quantification due to amplification failure. Since it is impossible to obtain a completely idealized nucleic acid template no matter what extraction method, although the quality of the nucleic acid template is related to the quality of the reagent, it cannot be completely resolved by the quality of the reagent.

Nucleic acid template problem In addition to the problem of the template itself, there is also the problem of inhibiting the action of Taq enzyme active components (such as certain proteins, ions, etc.) in the template solution, resulting in a decrease in amplification efficiency or even a failure of amplification. In this regard, some people advocate template purification, and the effect is more obvious. But another problem has appeared again, that is how to guarantee the recovery rate problem of purification. In conclusion, the nucleic acid template problem is an unavoidable problem that can only be minimized.

4. The quality of operators

There are many links in the PCR experiment, and the quality requirements for each link are very high, such as adding less, missing reagents, insufficient centrifugation, wrong cycle parameter design, degradation of RNA extraction, reverse transcription failure, etc. can cause results of false negatives. Therefore, PCR experiment operators are required to have good qualities, be able to strictly abide by the operating procedures, and be able to discover and solve problems keenly.

5. otherS

False negative results can be caused from the collection, transportation, and storage of samples in PCR experiments, and the periodic changes in the human blood system for pathogen detection (such as HBV) are also factors worth noting.

Anti-pollution strategies for PCR laboratories

The characteristic of PCR reaction is that it has great amplification ability and high sensitivity, but the troublesome problem is that it is easy to be polluted, and an extremely small amount of pollution can cause false positives.

Cause of pollution

1. Cross-contamination between specimens:

Specimen contamination is mainly due to the contamination of the container where the specimen was collected, or when the specimen was placed, due to the leaking of the container due to poor sealing, or the specimen sticking to the outside of the container, resulting in mutual cross-contamination; during the extraction process of the nucleic acid template of the specimen, due to the suction gun Contamination leads to contamination between specimens; virus samples can spread with aerosols or form aerosols, resulting in contamination among each other.

2. Contamination of PCR reagents:

The main reason is that during the preparation of PCR reagents, the sample gun, container, double distilled water and other solutions are polluted by the PCR nucleic acid template.

3. PCR amplification product contamination:

This is the main common contamination problem in PCR reactions. Because the copy amount of PCR product is large (generally 1013 copies/ml), which is far higher than the detection limit of several copies of PCR detection, so a very small amount of PCR product contamination can cause false positives.

Another form that is easily overlooked and may cause PCR product contamination is aerosol contamination. Aerosols can be formed when the air rubs against the liquid surface, and the reaction tube can be shaken violently during operation. When the cover is opened, the sample is sucked, and the sample gun is polluted repeatedly, the aerosol can be formed and polluted. It is calculated that an aerosol particle can contain 48,000 copies, so the pollution caused by it is a problem worthy of special attention.

4. Contamination of cloned plasmids in the laboratory:

In molecular biology laboratories and some laboratories that use cloned plasmids as positive controls or other positive samples, this problem is also relatively common, and the possibility of contamination is also high.

Pollution Monitoring

A good PCR laboratory should always pay attention to the monitoring of pollution, consider whether there is pollution and what causes the pollution, so as to take measures to prevent and eliminate pollution.

Controlled experiment:

1. Positive control:

The PCR positive control should be set up in the establishment of a PCR laboratory, which is an important reference sign of whether the PCR reaction is successful and meets the theoretical requirements. For the positive control, select samples with moderate amplification and good reproducibility, which are identified as the product after various identifications. If recombinant plasmid is used as the positive control, its content should be low rather than high (less than 100 copies). However, positive controls, especially recombinant plasmids and high-concentration positive samples, have a high possibility of contamination of detection or amplification samples. Therefore, when a certain PCR reagent is stable through self-use and the inspectors know it well, a strong positive control can be avoided in subsequent experiments, and a weak positive control can be used as a check for possible low-concentration virus samples. In addition, laboratories with mature conditions should choose positive reference products for indoor quality control products provided by a third party such as the Clinical Inspection Center of the Ministry of Health to monitor the entire reaction system.

2. Negative control:

Use known negative serum as a negative control, and qualified laboratories should use a third party such as a negative reference product for indoor quality control provided by the Clinical Inspection Center of the Ministry of Health to monitor the entire reaction system.

3. Repeatability test.

Ways to Prevent Pollution

1. Pollution Prevention

When performing PCR operations, operators should strictly follow some operating procedures to minimize or eliminate possible PCR contamination.

1. Division of operating areas: At present, ordinary PCR cannot achieve single-person, single-tube, and completely closed-tube operation. However, no matter whether single-person, single-tube can be achieved, the experimental operation is required to be carried out in three different areas. Pre-processing and post-processing are carried out in different isolation areas:

(1) Reagent preparation area, including preparation of reaction solution and storage of reagent consumables, etc.

(2) Specimen processing area, including the receiving and processing of specimens and the preparation of amplification templates.

(3) Amplification detection area, including PCR amplification detection, result analysis and report issuance.

(4) Each work area must be isolated to a certain extent, and the operating equipment must be dedicated and directional. The working flow of laboratory personnel and articles should be reagent consumable storage and preparation area → sample processing mixing area → sample preparation (nucleic acid purification) area → amplification detection area, and reverse flow is not allowed.

(5) Experimental supplies include experimental materials (reagents, specimens and consumables, etc.), experimental equipment (including containers, racks, lab coats, hats, masks, gloves, shoe covers, etc.), office supplies (all kinds of documents, recording paper, Pens, etc.) and cleaning utensils, etc., in order to facilitate identification, different work areas should use work clothes of different colors or with obvious distinguishing marks. When workers leave the work area, the work clothes specific to each area shall not be taken out.

(6) Improper cleaning methods are also an important cause of pollution, so laboratory cleaning should be carried out in the direction from the reagent storage and preparation area to the amplification detection area. Different experimental areas should use their own cleaning utensils to prevent cross-contamination.

2. Packing and dispensing reagents: All reagents required for nucleic acid extraction and PCR amplification should be prepared and packed on a ultra-clean workbench or negative pressure workbench (biological safety cabinet) equipped with ultraviolet lamps. All injectors and suction tips must be fixed in it.

3. Precautions for experimental operation: Although the residual contamination of the amplified sequence is mostly the cause of false positive reactions, cross-contamination between samples is also one of the reasons. Therefore, it is not only necessary to be cautious when carrying out the amplification reaction, but also to pay attention to all aspects of sample collection, extraction and amplification:

(1) Wear disposable gloves. If the reaction solution or serum sample is accidentally splashed, the gloves should be changed immediately.

(2) Disposable tips with filters should be used. It is strictly forbidden to mix them with the tips in various PCR areas. After the tips are used, the caps should be closed immediately, and they should not be exposed to the air for a long time to avoid aerosol pollution.

(3) Avoid splashing of the reaction solution. To avoid this situation when opening the reaction tube, centrifuge slightly before opening the lid to collect the liquid at the bottom of the tube. If it is accidentally splashed on the gloves or the table, the gloves should be changed immediately and the table should be wiped with 10% sodium hypochlorite.

(4) When operating multiple samples, they should be marked in advance, and then start the operation, so that the operation can be reduced, pollution can be avoided, and the accuracy of the reaction can be increased.

(5) The reaction tube to which the sample is added should be fitted with a cap and tightly closed.

(6) Negative and positive controls are set up during operation to verify the reliability of the PCR reaction and assist in judging the reliability of the amplification system.

(7) Use a replaceable or autoclavable sampler as much as possible. Since the sampler is easily contaminated by product aerosol or sample nucleic acid, it is best to use a replaceable or high-pressure sampler. If there is no such special sampler, at least the sampler should be dedicated during the PCR operation and cannot be used in cross-use.

(8) In the case of suspected samples, repeat or secondary hole experiments should be done when performing confirmation experiments to verify the results and draw conclusions carefully.

4. Personnel conducting blood nucleic acid testing must have a training certificate for blood nucleic acid testing, and have received training on laboratory biosafety and the manufacturer's pre-employment equipment operation, maintenance, and calibration.

5. For the control of outsiders in the laboratory, all personnel entering the laboratory, whether visitors, trainees, construction workers, etc., must abide by the laboratory regulations, strictly wear protective clothing, and strictly abide by the laboratory flow system.

6. Every nucleic acid testing laboratory personnel must have a strong anti-pollution awareness that is different from daily experiments.

2. Tracking of pollution sources

In case of accidental pollution, the following items should be analyzed one by one to eliminate the pollution.

1. Reagent contamination:

Set up negative and positive controls: it is beneficial to monitor the contamination of each component of the reaction system. When selecting a positive control, samples with weak amplification and good repeatability should be selected, because a strong positive control can produce a large number of unnecessary amplified sequences, which may become a potential source of contamination. If the reagent control is a positive result in the amplification result, it means that one or several reagents are contaminated. At this time, a batch of new reagents should be replaced for amplification. Different reaction tubes should be set up during amplification. Each tube contains a reagent to be tested. After the contaminated reagents are detected, they should be processed one by one immediately.

2. Environmental pollution:

In addition to eliminating the possibility of reagent contamination, if the reagent is found to be contaminated soon after replacing the reagent, if the precautionary measures are strict, it may be considered as environmental pollution. Common pollution sources in environmental pollution mainly include:

(1) Nucleic acid sample mixing and extraction equipment

(2) Biological safety cabinet

(3) Centrifuge

(4) Refrigerator door handles, freezer racks, door handles or lab benches, etc. At this time, the swab test can be used to find the suspected source of contamination:

(5) Aerosols.

If the exact source of pollution cannot be found after the above tracking experiments, the pollution may be caused by aerosols of PCR products in the air. At this time, the laboratory should be thoroughly cleaned or the experimental site should be replaced. If conditions do not permit, a new design should be made. primers (not related to the original primers).

3. Pollution treatment

1. Environmental pollution

(1) Dilute acid treatment method: Wipe or soak suspicious instruments with 1mol/L hydrochloric acid to depurinate residual DNA.

(2) Ultraviolet irradiation (UV) method: The ultraviolet wavelength (nm) is generally selected as 254/300nm, and it is enough to irradiate for 30 minutes. It should be noted that when choosing UV to eliminate residual PCR product contamination, 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 fragments above 500bp, and has little effect on short fragments. When UV is irradiated, the pyrimidine bases in the PCR product will form dimers, and these dimers can terminate the extension, but not all pyrimidines in the DNA chain can form dimers, and UV irradiation can also break the dimers . The extent of dimerization depends on the UV wavelength, the type of pyrimidine dimer and the sequence of nucleotides adjacent to the dimerization site. On irradiated long DNA strands, the number of dimerization defects is less than 0.065/base, and other non-dimeric photodamages (such as cyclobutane-type pyrimidine complexes, thymine glycol, DNA interstrand Intra-strand cross-links and DNA breaks, etc.) can terminate the extension of Taq DNA polymerase. The number of these sites is comparable to that of dimer sites. If these sites (0.13/base) are randomly distributed on the DNA molecule, there will be 32 damage sites on the DNA molecular chain of a 500bp fragment, then, there will be at least one damage site in each molecule of 105 such molecules . Conversely, if a 100 bp fragment has only 6 lesions per strand, many of the 105 copies of the molecule will not have any lesions. This is why UV irradiation has a certain fragment length limitation.

2. Reaction solution contamination

Can be handled in one of the following ways:

(1) DNase I method: Add 0.5 U DNase I to the PCR mixture (without template and Taq polymerase), react at room temperature for 30 min, heat inactivation, and then add template and Taq polymerase for normal PCR amplification. The advantage of this method is that no knowledge of the sequence of the contaminating DNA is required.

(2) Endonuclease method: Select an endonuclease that recognizes 4 bases (such as Msp I and Taq I, etc.), and several kinds can be selected at the same time to overcome the defect that one enzyme can only recognize a specific sequence, and it can be used at room temperature After 1 h, heat inactivation was performed for PCR.

(3) Ultraviolet irradiation method: the PCR mixture solution without template and Taq polymerase is subjected to ultraviolet irradiation, and the precautions and methods are the same as the above-mentioned UV irradiation method.

(4) g-ray radiation method: 1.5 kGy radiation can completely destroy 0.1ng of genomic DNA, 2.0 kGy can destroy 104 copies of plasmid molecules, and 4.0 kGy still does not affect PCR, but higher than this limit will reduce the efficiency of PCR amplification. Primers can be irradiated without affecting PCR, and g-rays generate free radicals through ionization of water to damage DNA.

3. Uracil glycosidase (UNG) method

Because the decontamination effect of UV irradiation is not effective for fragments below 500bp, and the PCR amplification fragments used for clinical detection are usually about 300bp, so the preventive effect of UNG is increasingly valued and affirmed.

(1) Principle: Use dU instead of dT in the PCR product or primer. The dU-based PCR product is incubated with UNG, because UDG can cleave the N-glycosyl bond between the uracil base and the sugar-phosphate backbone, and can remove dU prevents the extension of TaqDNA polymerase, thereby losing the ability to be reamplified. UNG has no effect on dU-free templates. UNG eliminates uracil from single- or double-stranded DNA, but has no effect on uracil and single uracil molecules in RNA.

(2) dUTP method: replace dTTP with dUTP, so that a large amount of dU is mixed into the product. Before performing PCR amplification again, the residual contamination of the PCR product can be eliminated by treating the PCR mixture with UNG. Since UNG can be inactivated by one step of denaturation in the PCR cycle, it will not affect the new PCR products containing dU.

(3) dU primer method: When synthesizing primers, dT is replaced by dU, so that only the 5′ end of the PCR product has dU. After UNG treatment, the primers lose their binding sites and cannot be amplified. For the amplification of long fragments (more than 1-2kb), the efficiency of the dUTP method is lower than that of dTTP, and the dU method can overcome this shortcoming. For dU primers, it is best to design dU at the 3' end or near the 'end. This method can only be used for the treatment of reagents other than primers.

(4) Advantages: contamination from any source can be removed; UNG treatment and PCR amplification can be carried out in the same reaction tube; since a large amount of dU exists in the amplification product, contamination sources can be completely eliminated.

(5) It should be noted that the dUTP-incorporated DNA should not affect any operation of the product. When cloning the PCR product, it should be transformed into UNG- (UNG-deficient) E. coli recipient bacteria, otherwise the transformed product will be destroyed by the recipient bacteria. UNG digested.

4. Daily decontamination measures include but are not limited to the following:

(1) Clean the surface with 10% sodium hypochlorite or 75% ethanol, including the experimental operation table (daily), storage refrigerator (at least once a week), instrument surface (daily), etc.;

(2) After the test, long-term ultraviolet irradiation experiment operation table and other surfaces; 1 hour of mobile ultraviolet car 60-90CM irradiation table, more than 4 hours of indoor irradiation with fixed ultraviolet lamp;

(3) High-pressure disinfection of high-pressure injectors; disinfection of scissors, tweezers and other supplies (once a week);

(4) The laboratory is ventilated for more than 2 hours (after daily experiments), and the windows must be equipped with screens.


Treatment and verification principles of nucleic acid residual contamination in the laboratory

1. Terminate the experiment:

Once pollution occurs, it is time-consuming and cumbersome to search for the source of pollution around the laboratory, so the prevention of pollution is the most important thing. But if contamination occurs, the experiment must be stopped until the source of the contamination is discovered and removed, and the results of the experiment must be invalidated.

2. Verification of contamination removal:

Starting from nucleic acid extraction, test 15-20 pure water samples step by step according to the procedure, and observe whether there are positive reaction results. If so, it means that there is still contamination in the laboratory, and the laboratory must be cleaned until all water samples are tested negative before the laboratory can be reopened.

Generally speaking, the factors that most affect the experimental results are reagents, environment, equipment, and personnel, among which personnel have great variability. Combining the long-term experimental data collation and the analysis of quality control charts, we will find more problems and solve them. We will continue to persist in all good ones, and resolutely abandon bad habits. Only with a serious work attitude and prudent operation Only by completing daily normal testing experiments and eliminating or reducing the occurrence of false positives can the normal development of blood screening experiments be guaranteed.