Multiplex PCR is a technique established on the basis of common PCR to detect a variety of bacterial molecular biology that can amplify multiple target fragments at the same time, which utilizes multiple pairs of specific primers to amplify at the same time, improving the amplification efficiency and saving costs. The advantages of this technique are high speed, high accuracy and simple operation, which is especially suitable for clinical, environmental and food testing.
Principle
Multiplex PCR is a new technology improved on the basis of conventional PCR, and its basic principle is the same as that of conventional PCR, which is to achieve the amplification of the target fragment through polymerase chain reaction.
When the DNA double strand is at a high temperature of 90 ℃, the double stranded DNA will be deconvoluted into single stranded DNA; when the temperature is lowered to about 60 ℃, the primers will be combined with the specific target sequences; when the temperature is raised to 72 ℃, which is the optimal temperature of DNA polymerase, the extension of the single stranded DNA can be completed through the principle of base complementary pairing on the basis of a variety of dNTPs substrate, and the paired synthesis of DNA double strand will be completed finally. The final synthesis of DNA double strand is accomplished through the principle of base complementary pairing.
The difference between multiplex PCR and conventional PCR is the number of primer pairs added to the same reaction system. Multiplex PCR can specifically amplify multiple target DNA fragments. The composition of the reaction system and the conditions of the PCR cycle need to be optimized to ensure the simultaneous amplification of multiple DNA fragments. Theoretically, the number of primer pairs can be unlimited as long as the conditions for PCR amplification are appropriate, but the number of primer pairs that can be amplified in practice is limited due to a variety of conditions (∼1,000 pairs).
Operation method
Bacterial multiplex PCR rapid test
Principle
Multiplex PCR is a new technology improved on the basis of conventional PCR, and its basic principle is the same as that of conventional PCR, which is to achieve the amplification of the target fragment through polymerase chain reaction. When the DNA double strand is at a high temperature of 90 ℃, the double stranded DNA will be deconvoluted into single stranded DNA; when the temperature is lowered to about 60 ℃, the primers will be combined with the specific target sequences; when the temperature is raised to 72 ℃, which is the optimal temperature of DNA polymerase, the extension of the single stranded DNA can be completed through the principle of base complementary pairing on the basis of a variety of dNTPs substrate, and the paired synthesis of DNA double strand will be completed finally. The final synthesis of DNA double strand is accomplished through the principle of base complementary pairing. The difference between multiplex PCR and conventional PCR is the number of primer pairs added to the same reaction system. Multiplex PCR can specifically amplify multiple target DNA fragments. The composition of the reaction system and the conditions of the PCR cycle need to be optimized to ensure the simultaneous amplification of multiple DNA fragments. Theoretically, the number of primer pairs can be unlimited as long as the conditions for PCR amplification are appropriate, but the number of primer pairs that can be amplified in practice is limited due to a variety of conditions (∼1,000 pairs).
Materials and Instruments
Target strains (Staphylococcus aureus for example), Tryptone, Bacterial DNA Extraction Kit, LB Liquid Medium, LB Solid Medium; PCR Thermal Cycler, PCR Amplifier etc. Move 1. Primer design: Primers were designed for genes with sequence length above 500 bp. Firstly, use the Primer software, then analyze the primers by Oligo software and BLASTN algorithm, choose the primers with less dimer, no hairpin structure, Tm value around 60 ℃ and low homology with non-DNA as the specific primers of the gene, and send them to the company for synthesis. It is better to carry out primer evaluation experiments. 2、Extraction of DNA of the target bacterial strain: take up the sample homogenate, centrifuge at 5,000 r/min and discard the supernatant, then extract the genomic DNA of the sample by referring to the bacterial DNA extraction kit. 3、Determination of DNA concentration and purity: when the ratio of 260 nm/280 nm is between 1.8-2.0, it indicates that the extracted genomic DNA is of good quality. 4. Multiplex PCR: The extracted genomic DNA was used as a template for multiplex PCR reaction with the designed primers (SU4, smal, clfA). 25 μL of multiplex PCR reaction system was as follows: 2.5 Μl 10 X PCR Buffer, 1.0 μL MgCl2 (4 mM), 2.5 μL dNTP Mixture (2.5 mM each), 0.5 μL Taqqa Mixture (2.5 mM each), 0.5 μL Taqqa Mixture (2.5 mM each), 0.5 μL Taqqa Mixture (2.5 mM each). The PCR reaction was performed with 0.5 μL Taq enzyme (5.0 U), 2.0 μL DNA template, primers (10 umol), 2.6 μL of SU4, 0.5 μL of smal, 1 μL of clfA, and 25 μL of ddH2O. 5, PCR reaction program: ① 94 ℃ 5 min; ② 94 ℃ 30 s; ③ 58 ℃ 30 s; ④ 72 ℃ 45 s; ⑤ 72 ℃ 10 min. ② - ④ step was repeated 30 times. 6. Configure 1.5% agarose gel and use electrophoresis to detect the multiplex PCR reaction products. 7. Stain in EB solution for 10~15 minutes and put it under the gel imager to check the results of electrophoresis bands. Caveat 1. when the ratio is greater than 2.0, it indicates that the extracted genomic DNA contains a high amount of RNA, and a certain amount of RNase should be added to remove RNA from the solution. 2. the length of the target fragments is the same, and smaller fragments are always preferred for amplification. 2. the length of target fragments varies, and smaller fragments are always preferred for amplification. 3. the optimal PCR conditions for each primer are as follows3. the optimal PCR conditions for each primer pair are different, with longer fragments requiring longer extension times. In order to ensure the consistency of the final amplification yield, when optimizing the reaction conditions, choose the amplification conditions that favor the larger fragments as much as possible. 4. In the multiplex PCR system, there is a positive relationship between the amount of primers and the length of the amplified target fragment, i.e., the longer the amplified fragment is, the more primers are required, and the interaction between the primers will lead to poor amplification. 5. The optimal concentration of DNA polymerase is 2 U per 25 uL of reaction volume, which is slightly adjusted according to the amplification results. 6. High salt concentrations make it difficult to denature and unstrand long-fragmented amplification products. As a result, long fragment products are better amplified at low salt concentrations, while short fragment products are better amplified at high salt concentrations. The concentration of PCR buffer can be increased appropriately, or the K+concentration to 70~100 mM. 7. Different bacterial types and sample types may require different extraction and amplification methods, which need to be selected according to the specific situation. Meanwhile, when performing PCR amplification reaction, it is necessary to pay attention to the control of reaction conditions and reduce contamination to ensure the accuracy and reliability. For more product details, please visit Aladdin Scientific website.