PCR technology

PCR technology with a high degree of specificity, selectivity, sensitivity, and fast, simple and easy automation, in clinical work in wide attention and reasonable application. At present in many of our primary health care units have been carried out PCR application of the research. PCR technology is very simple, very clear principle, conditional units can be implemented more smoothly, but after all PCR technique is a new thing, subject to many conditions factors, it is not easy to do better. PCR technology applications in the encounter in the course of this or that issue, even more will be completed with the fact that the opposite result. To enable us to smoothly carry out PCR work, and better tools to play the role of PCR technology, we work under the PCR years of lessons learned, the PCR amplification process of some of the problems often appear with a brief summary, available to the general scientific research Workers as a reference, Paozhuanyinyu, welcomed the inadequacy of the correction. A false positive amplification Sometimes in the course of the experiment will be met by all the samples tested positive, and electrophoresis of PCR products with uniform brightness, which is amplified when the system is polluted the most typical of a performance, need to carefully inspect and remove sources of pollution, the general should be Start the following steps: ⒈ reagents pollution: Reagents provided by the manufacturer or a component in the factory or the transport, storage process of being contaminated. Detection method can be reagent kit brochures will be installed at a direct place in the PCR-amplified (without the negative control, and adequate distilled water), will be simple and effective reagent to judge whether the contamination. ⒉ laboratory contamination: This is the most common sources of pollution, because of PCR technology in a few hours in the template will be some specific amplification to the millions of times more likely to result in the expansion of mandrax electrophoresis process of pollution, such as pipettes, console With the evaporation or by the formation of aerosol pollution and the whole laboratory. Amplified products is the most effective templates, once the pollution of their pollution levels are more serious. Judgement: the experiment could be the most crucial steps such as packaging reagents, sample processing transfer to a new environment or transferred to the ultra-clean work Taichung completed. Of course, the use of pipettes, Tsui suction tube (centrifuge tube) should be completely replaced. Solution: laboratory contamination of PCR is the most vulnerable in the course of the phenomenon, but once the pollution, eliminate sources of pollution also extremely difficult, often takes the entire laboratory and experimental equipment cleaned thoroughly dealt with, so it should be a prevention-oriented, experimental In strict compliance with the basic requirements of sample processing and amplification and electrophoresis product should be separated as much as possible, preferably in two rooms. Amplified by the pre-treatment with pipettes and after the sample was amplified by the Pipettes should be strictly separated, not interchangeable. PCR room should maintain good ventilation, cleaning, preferably exclusive. ⒊ pollution sampling: In the course of the experiment, and sometimes there will be a number of positive results of the high rate, and a number of positive results and rate of decline in many, so repeatedly, a phenomenon largely because of pollution caused by sampling, and generally speaking the formal reagent manufacturer, its agent The factory to go through strict quality inspection, grant, award, in particular the differences are very small, so obviously there will not be repeated, this phenomenon was mainly due to the collection of samples or suction tube caused by pollution. PCR amplification is very sensitive, and general laboratory test tube by the repeated use of the injection can not be washed way to remove trace DNA, trace DNA of these will become PCR template, a positive PCR result, the degree of contamination sample test tube Different, so there Mandrax Mandrax high-low phenomenon solutions recommended a suction tube and Tsui sampling. Second, the false negative amplification: If successive negative results are amplified, or known positive samples appear negative PCR result, the general view that this is false negative, there is this phenomenon for several reasons: ⒈ equipment failures: A full-Yam results, first of all to consider whether the normal operation of equipment. Correct judgement of the equipment working conditions, ruled out false negative is the basis for other reasons, the equipment is functioning properly PCR amplification Buju one of the most crucial. Judgement see if the equipment is normal, we must first of heating temperature of the accuracy of the volatility of the Chinese request, the difference between whether the hole to meet the requirements, PCR amplification of equipment are often heated and the temperature difference between the requirements of a high Accuracy, if too much error, it is bound to arise false negative. It should be noted that the letter can not be famous, we often found that some brand-name machines temperature differences as high as second over. ⒉ reagent failure or invalid: Whether the normal understanding of machinery, the reagent can detect. We must first understand whether the agent over the period to test whether the method of storage requirements, if the kit in the period to which the storage method is correct, then they should be carefully, carefully determine whether the reagent is invalid or failure reagents reagents. The positive samples can be known, or kit provided by the positive control, expanded operations in strict accordance with a statement, and then amplified by double-distilled product diluted 1,000 times, as a template for the second time amplification, judge the results, if it is Negative note reagent is invalid or failure, if the positive results of the following methods available then determine the sensitivity of reagents. ⒊ reagent sensitivity: Sometimes the positive test for low rate, the so-called partial-positive rate is the rate that a false negative result, this phenomenon often associated with the following three factors: But the quality of reagents ① Commissioner; ② operation was not standardized; ③ subjective criteria unscientific. The reagent is correct quality and operation of these two points is relatively easy to check and a reasonable improvement. Focus to mention here: there often encounter some unscientific concept, we used to use some samples should be negative, some samples should be positive, or simply to positive rates. The concept of subjective judgement whether the reagent qualified This is a very unscientific and subjective reasoning. If the determination of serum HBV, can not be used abstract should be a positive rate of 60% or 20% of such a concept to judge, the same can not "two pairs of semi-" the result of judgement "PCR results," This is because the "two On a half "and" PCR "are two totally different detection method, the results of the significance of the difference between its medical personnel for the information provided is very different and more by the use of the" two pairs of semi-"Reagents Whether as a quality evaluation standard test yet to be determined. The world's more recognized standards for Abbott's only "two pairs of semi-" reagents, domestic "two pairs of semi-" According to the Ministry of Health reagent checks show that the pass rate was 50 percent, how can you be assessed as a quality standard? Reagents sensitivity of the evaluation criteria should use the following method of scientific evaluation, the first sample selection criteria such as HBV sequence plasmid, the standard positive serum, such as tuberculosis bacteria use the appropriate medium such as serum and sputum, and so on a limited dilution 1:10,1:100,1:1000,1:10000 so gradually raise the price of its dilution of the sensitivity of reagent is generally believed that if the serum HBV positive standards with normal serum diluted 10,000 times more, per milliliter of TB In about 100 still measured positive results, the sensitivity of the reagent is sufficient to determine the sensitivity of reagent is sufficient to determine the sensitivity of reagents be judged after the detection reagent coverage, because pathogenic micro-organisms have different type or variation Strain, qualified reagents should not be Lou Jian. Finally kits to determine the opposite sex, the general laboratory can be used by different pathogens Manual operation of the sample to determine its expansion to other pathogens, also have a positive, the specificity of the accuracy of what can be achieved, after more than three areas of Scientific research will be able to judge the quality of reagents. Third, the amplified products tailing Sometimes amplified products electrophoresis after one fluorescent Mission (Smear phenomenon), or a series of bands, these kinds of phenomena with the following major factors: ⒈ amplification system flawed: This phenomenon shows that a non-specific amplification, and a non-specific amplification and amplification system in the concentration of magnesium ions, with concentration, DNTP is closely related to the concentration required serious adjustment with a view to avoid such a phenomenon. It is also appropriate to improve the annealing temperature, thereby enhancing amplification specificity. ⒉ imperfect template approach: The principle of PCR Although very simple, but the rational application of this technology is extremely complicated, such as the PCR DNA polymerase one of the major components of the impact of many factors, what is the role of these factors in the polymerase, the mechanism so far Still unclear, so is wrong with the improper handling of samples may inhibit amplification, may also lead to non-specific amplification. General secretion samples, mainly from exfoliated cells, to avoid collecting too many Nongxing secretions, and sputum samples must fully liquefaction, and so on. ⒊ primer design unreasonable: Primer design should be guided by several principles. First, the general primer should be composed of 15-30 months BP; Second, the primer in the base should be randomly distributed, the same can not have a bunch of other bases or not a common structure; third, GC base content should be 45 -- About 55%; Fourth, the two primers in the 3 'non-identity can not be there. To which primers and gene homology between the group is a non-specific amplification of the main reasons, we generally in the design of primers, often only aware of their genome sequence in a certain period, so we must make full use of such information ingenious design To a reasonable primer, and through repeated comparison, confirmed that the final to determine its ability to detect. 4, single-product of electrophoresis and fluorescence with more general with fluorescent PCR amplification, the PCR results of the judgement the most simple way is to agarose gel electrophoresis, bromine B amylin staining, 320 nm in the UV excitation observed Fluorescent strip, if there are clear and specific amplification product will be flat in electrophoresis expected a clear position in the fluorescent-band amplification system in accordance with the standard preparation of the response, with its concentration in the 0.1-0.5 mM , The general circulation by 30 after the expansion, there is still a considerable number of free primer exists, so there is a slab electrophoresis about 20 BP (electrophoresis faster), fluorescent light with the primer, so each electrophoresis Have two Ying-zone, in front of one, each of the points have everything with the primer. Primer design very important, because a large genome DNA sequence, in addition to the specific amplification, are often very easy to produce non-specific product. If the primer with the genome of the existence of a broader identity, then there will be serious non-specific expansion of mandrax, not only with the specific primer amplification regional integration, but also a series of regional and other non-specific binding, and the choice of primers, , And often have to consider sensitive issues, especially the clinical test kit, less able to detection of pathogens to a few, we mostly used in the pathogen genome duplication in the DNA sequence design primer, if they repeat not strictly homologous , On the possible length of the amplified products and a multi-band amplification. Pathogenic variant, with the foregoing, we often use the high sensitivity of a repeat of the gene as a primer design section, in some pathogens, such as tuberculosis in the treatment process, will be a serious distortion, including its Genetic material changes in the chromosome deletion, ranging from digital exchange, such as the insertion of other sequences. If these deficiencies and a range of the exchange is in our choice of amplified sequence, will also appear different length of the amplified products, resulting in a number of amplified products with. PCR gene amplification technology simple, application of the increasingly widespread, but its impact on many factors, in the course of practice is now often this or that issue, not even the expected results, or appear unable to explain the phenomenon. It is true that a large number of scientific workers in recent years with concerted efforts, made many valuable experiences, so that this technology are maturing, after all, PCR is the only one in recent years the emergence of new technologies, many of the problems is still not very good To explain or resolve. With the letter to the PCR in-depth study, the application of the PCR process of constantly sum up experience, PCR this new technology for human development will make