Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 150 polymerase (such as Taq enzyme), so that the target sequence multiplies exponentially. The PCR reaction is highly specific, and amplification bands are generated only when there is a viral nucleic acid matching the primer in the sample, thereby achieving qualitative detection of the virus. This method does not require high experimental conditions, and a conventional PCR instrument can complete the amplification process. The results of RT-PCR are usually observed by agarose gel electrophoresis to see the size and presence of the amplified band, and can be compared with the positive control to determine whether the virus exists. Due to its advantages of high sensitivity and strong specificity, RT-PCR is widely considered to be one of the "gold standard" methods for plant virus detection (Zhang et al., 2016). 3.2 Sensitivity and specificity analysis RT-PCR has extremely high sensitivity due to its high amplification efficiency and can detect extremely low concentrations of viral nucleic acids in the sample. Studies show that the detection sensitivity of RT-PCR on potato viruses can reach the pg order, which is hundreds to thousands of times higher than traditional ELISA. RT-PCR is highly specific and depends on the base complementary pairing of primers and templates. If the primers are designed properly, nonspecific amplification will be difficult to produce even if the gene sequences are similar between different viruses (Zhang et al., 2016). On the one hand, since the concentration of PCR amplification products is very high, it is easy to cause aerosol contamination, it should be strictly divided and a negative control should be set up in each batch of reactions to monitor the contamination. On the other hand, plant extracts may contain substances that inhibit PCR, such as polyphenols and polysaccharides, which need to be removed during RNA extraction and purification, or add bovine serum albumin (BSA) and other factors to relieve inhibition in the PCR system. In addition, internal standard controls (such as plant endogenous genes) can be added to the detection system to verify whether the PCR reaction is running successfully. 3.3 Interpretation of test results and quality control measures The detection results of RT-PCR are usually presented in the form of electrophoretic bands and need to be explained in conjunction with appropriate controls. Positive controls generally use positive templates known to contain target viruses, and their target bands show that the system responds normally; negative controls are reactions without templates (or healthy plant extracts), and no amplified bands should appear. The sample had a band of the same size as the positive control and the fluorescence intensity was sufficient, so it was judged to be positive for the virus; if there was no band, it was negative. In terms of quality control, in order to prevent false positives, PCR steps must strictly follow the principles of sterile operation and partitioning: extraction, preparation, amplification and electrophoresis should be carried out in different areas, and disposable filters should be used to avoid aerosol contamination. Once contamination is suspected, primers or enzyme preparations can be replaced and DNA removal treatments (such as UV irradiation, etc.) of the laboratory environment (Lee and Rho, 2015). For high polysaccharide/polyphenol samples, adsorbed impurities such as PVPP can be added during extraction, or additives can be added to PCR to improve amplification. Positive plasmids with known concentration gradients should also be included in the experiment to evaluate amplification efficiency and sensitivity. When detecting multiple viruses, primers need to avoid interference and competition between each other, and parallel detection of the multiple PCR system can be used (Rashid et al., 2021). 4 Real-Time Fluorescence Quantitative PCR (qRT-PCR) 4.1 Quantitative detection principle and primer/probe design principle Real-time fluorescence quantitative PCR (qPCR or qRT-PCR) is the real-time monitoring of product accumulation through fluorescence signals during PCR amplification, thereby achieving quantitative analysis of the number of initial templates (Zhou et al., 2019). Unlike traditional PCR, qPCR uses fluorescent dyes (such as SYBR Green) or specific fluorescence probes to indicate the production of amplification products, measuring fluorescence intensity per cycle and drawing an amplification curve. When the fluorescence signal reaches the preset threshold, the number of cycles (Ct value) is negatively correlated with the initial template amount, and the copy number of viruses in the sample can be calculated based on the standard curve. The principle of qRT-PCR for RNA viruses is one-step or two-step RT-PCR combined with fluorescence detection. In primer and probe design, higher
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