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Since PCR technology was invented by Mullis in 1985, it has been widely used in the detection of nucleic acid molecules. But obviously, it can only be qualitative analysis but not quantitative, which has become a major problem that plagues scientific researchers. Just 5 years later, there were some preliminary attempts, and good results were achieved.
Pang et al. used a method of molecular imaging after PCR for quantification [1]. The PCR raw material ATP was labeled with 32P, incorporated into the product, and analyzed with auto-imaging technology (Fig 1). The accuracy is sufficient to distinguish the difference of twice the concentration. This method is relatively cumbersome to operate and involves radioactive labeling; in addition, its detection method is still slightly rough, only considering the optical density value of the band, which belongs to the category of semi-quantitative, and it also needs to control the number of amplification cycles in the exponential phase ( Within 30). Although this method has not been widely used, it has taken a breakthrough step.
Fig 1. Autographic detection of genomic DNA and HIV-1 DNA
A more widely used quantitative method appeared in 1991, called Quantitative Competitive PCR (QC-PCR) [2]. The principle is to add a known concentration of internal reference template, namely competitor, to the sample, which has the same amplification efficiency as the target in the sample, so that the ratio of C to T after the amplification is completed (not necessarily in the exponential phase) It is the same as before amplification, and the C:T value after amplification can be obtained by electrophoresis analysis, so as to deduce the number of targets before amplification (Fig 2). The accuracy and sensitivity of this method have been greatly improved. When applied to the detection of HIV-1, the sensitivity can reach 100 copy per reaction [3]. But its shortcomings are also obvious. In addition to the slightly cumbersome operation, how to design a competitor is the biggest difficulty.
Fig 2. Schematic diagram of QC-PCR principle (Takara Competitive PCR Guide)
Although the previous two methods are not satisfactory, they provide a correct solution for the birth of qPCR-how to establish a relationship between the PCR product concentration and the initial template concentration. In 1996, the publication of Real Time Quantitative PCR marked the birth of qPCR [4]. It introduces a fluorescent-labeled hydrolysis probe, and collects the fluorescent signal after the extension of each PCR cycle. The number of cycles required to reach the exponential amplification phase (Ct value, which will be standardized to the Cq value after normalization) and the template The initial concentration establishes a relationship (Fig 3), so that the number of targets in the unknown sample can be quantified (the principle will be described in detail in the next section).
Fig 3. Real-time detection of fluorescence signal and establishment of standard curve
This article examines the precision of this method in detail and shows that it is still highly reproducible for different sample processing methods. The article also mentions that qPCR has three advantages compared with the previous two methods: 1. The detection is performed in the system without the need to open the lid and run electrophoresis after PCR, which reduces the possibility of product contamination; 2. It supports housekeeping gene as a calibrator without adding internal control, and multiple detection is possible; 3. Short detection time and sample access The amount is high. This article has been cited more than 7000 times (Google Scholar), and its groundbreaking and classic character is beyond doubt.
Since this article, various technological innovation-type articles have sprung up. Philip S et al. creatively used probe technology to make melting curves (Fig4) to distinguish different variants of hemochromatosis genes [5]; Jacqueline et al. used qPCR technology to simultaneously detect four retroviral nucleic acids [6], and multiple detection became more mature. Yolanda et al. reviewed the application of qPCR in copy number determination, RNA expression and allele discrimination [7].
Fig 4. Fluorescence resonance energy transfer (FRET) technology for dissolution curve analysis
Michael et al. described the method of combining qPCR with antigen and antibody to detect trace amounts of protein [8], replacing the traditional Biotin-Streptavidin biological system with PCR (Fig. 5). The signal amplification function is more advantageous. The sensitivity of protein detection is improved by two to three orders of magnitude compared with traditional ELISA. Although this article is not the first, it makes the method more systematic and perfect. In 2001, Livak proposed to use the familiar 2-△△Cq to process gene expression data[9], the whole process was more refined and simplified; later Pfaffl and Vandesompele improved the calculation methods based on different experimental conditions [10][11 ], the application conditions are more realistic.
Fig 5. The principle difference between Real-time Immuno PCR and traditional ELISA
Until recently, more nucleic acid quantification technologies based on qPCR have been published. Yanan Du et al. described a nucleic acid multiplex detection (EPFS System, see Fig 6) achieved by emulsification PCR combined with spectral detection [12]. The sensitivity, specificity and reproducibility were evaluated in detail. Tested in.
Fig 6. Schematic diagram of emulsification PCR technology (EPFS) for fluorescence molecular spectroscopy
It has been 23 years since qPCR came out, and today, with the rapid development of science and technology, it has been regarded as an "old technology job". However, we can still derive nourishment from these old technical activities, intensively cultivate or find new ways; this is what we mean when old trees bloom new flowers. In the future, the combination of DNA fluorescence molecular detection, microfluidic technology and even flow cytometry will surely make qPCR a different style.
references
1. Pang S,Koyanagi Y, Miles S, et al. High levels of unintegrated HIV-1 DNA in braintissue of AIDS dementia patients[J]. Nature, 1990, 343(6253): 85.
2.Becker-Andre, M. Quantitative evaluation of mRNA levels. Method Molecular Cell Biology. 1991. 2:189-201
3. Piatak,Michael, et al. "High levels of HIV-1 in plasma during all stages ofinfection determined by competitive PCR." Science 259.5102 (1993):1749-1754.
4. Heid C A,Stevens J, Livak K J, et al. Real time quantitative PCR[J]. Genome research,1996, 6(10): 986-994.
5. Bernard,Philip S., et al. "Homogeneous multiplex genotyping of hemochromatosismutations with fluorescent hybridization probes." The American journal ofpathology 153.4 (1998): 1055-1061.
6. Vet,Jacqueline AM, et al. "Multiplex detection of four pathogenic retrovirusesusing molecular beacons." Proceedings of the National Academy of Sciences96.11 (1999): 6394-6399.
7. Lie Y S,Petropoulos C J. Advances in quantitative PCR technology: 5′nuclease assays[J]. Current Opinion in Biotechnology, 1998, 9(1): 43-48.
8. Adler M,Wacker R, Niemeyer C M. A real-time immuno-PCR assay for routine ultrasensitivequantification of proteins[J]. Biochemical and biophysical researchcommunications, 2003, 308(2): 240-250.
9. Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-timequantitative PCR and the 2− ΔΔCT method[J]. methods, 2001, 25(4): 402-408.
10.Pfaffl, Michael W. "A new mathematicalmodel for relative quantification in real-time RT–PCR." Nucleic acidsresearch 29.9 (2001): e45-e45.
11. VandesompeleJ, De Preter K, Pattyn F, et al. Accurate normalization of real-timequantitative RT-PCR data by geometric averaging of multiple internal controlgenes[J]. Genome biology, 2002, 3(7): research0034. 1.
12. Du Y, ZhaoX, Zhao B, et al. A novel emulsion PCR method coupled with fluorescencespectrophotometry for simultaneous qualitative, quantitative andhigh-throughput detection of multiple DNA targets[J]. Scientific reports, 2019,9(1): 184