Quantitative polymerase chain reaction (Q-PCR) for detection of sugarcane streak mosaic virus

Abstract

Conventional PCR is a reliable method for detecting SCSMV. The availability of quantitative PCR (qPCR) can increase the power of PCR as detection method due to its high sensitivity and ability to quantify the template DNA in the reaction mixture. The study was aimed to validate the qPCR method for detection of SCSMV, and to develop detection methods for virus indexing program in seed canes production. qPCR method developed in this study was relative-qPCR with the regression equation of Y = -7,255 * log X + 11.752 for the SCSMV standard curve, so the value of X = 10 (-0.138 * Ct + 1,620). Ct value is inversely proportional to the concentration of cDNA. The greater the Ct value, the lower the concentration of cDNA in the sample. It was proved that qPCR method is more sensitive compared to conventional PCR, because it was able to detect SCSMV in samples not detected by conventional PCR methods. The relation between the Ct value and the incidence and severity of the disease on the field was assessed using field samples for qPCR. It was shown that Ct value and virus concentration did not have relation to the incidence and severity of the disease in the fields.

Construction of Very Low-Cost Loop Polymerase Chain Reaction System Based on Proportional-Integral-Derivative Temperature Control Optimization Algorithm and Its Application in Gene Detection

“…Polymerase chain reaction (PCR) technology developed in 1983 by Kary Mullis has been a well-known method and has been used widely in biological and medical invention. − By applying this technology, the natural replication process of DNA and specific DNA molecular fragment amplification in vitro could be obtained, which has been popularized and applied in the detection of various pathogenic microorganisms and parasites, especially in the point-of-care testing (POCT), such as disaster rescue, , quarantine, , and plant disease monitoring. , There exist three stages in a PCR system, namely, denaturation, anneal, and extension. , When the reagent is heated up to approximately 95 °C, the double-stranded DNA would be dissociated into a single one and combined with the primer in the next round of reactions, which is called as the denaturation of the template DNA stage. While the system’s temperature dropped to presumably 55 °C, the template of the single-strand DNA combined with the primer based on the principle of complementary base pairing, which is called as the annealing of the template DNA and the primer stage.…”

“… 1 − 3 By applying this technology, the natural replication process of DNA and specific DNA molecular fragment amplification in vitro could be obtained, which has been popularized and applied in the detection of various pathogenic microorganisms and parasites, 4 especially in the point-of-care testing (POCT), such as disaster rescue, 5 , 6 quarantine, 7 , 8 and plant disease monitoring. 9 , 10 There exist three stages in a PCR system, namely, denaturation, anneal, and extension. 11 , 12 When the reagent is heated up to approximately 95 °C, the double-stranded DNA would be dissociated into a single one and combined with the primer in the next round of reactions, which is called as the denaturation of the template DNA stage.…”