MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

Abstract

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

“…Specific concerns are related to false negativity as a result of decreased probe binding efficiency or complete binding failure due to mutations in the corresponding template regions34567891011121314. Hence, even if the specific amplicons are generated, the assays remain non-fluorescent, thus suggesting no amplification in a TaqMan qPCR setting.…”

“…Further, identically labelled FAM probes enable pre-existing internal controls to be used without the necessity of re-labelling. Finally, they provide the option of advanced analyses14, and their use is cost-effective.…”

“…Mismatches in the probe-binding region decrease or completely eliminate the probe binding, leading to false negativity. This is an important and frequently reported issue in diagnostic virology34567891011121314 that is faced in the detection of highly variable viral nucleic acid (NA) templates. Therefore, it is crucial to design novel approaches to improve the inclusivity and to reduce the probability of false negative results of diagnostic TaqMan qPCR assays.…”

A Rapid Diagnostic for Detection ofAphelenchoides besseyiandA. fujianensisBased on Real-Time PCR

“…In general, although hydrolysis probes have greater specificity, they are susceptible to template mutations. Thus, mutations in the probe hybridization site may reduce efficiency and sensitivity of the qPCR assay or may block the probe binding completely, leading to false-negative results (Nagy et al 2016;Yao et al 2006). Analysis of intraspecific variability in A. fujianensis detected the presence of mutation at the probe site of a single cloned individual ( Supplementary Table S2) but this did not affect the efficiency of amplification of the target genomic DNA from single nematodes (Table 5).…”

“…Interpretation of Cq $ 35 is often a challenge in qPCR assays because they may indicate the detection of low amounts of target DNA or be caused by poor amplification efficiency, as much as can be derived from nonspecific probe hydrolysis or even probe degradation (Nagy et al 2016). To underpin the diagnostic and provide phytosanitary laboratories with additional provenance to support their findings, we propose the use of LDA and tree classification analyzes to generate a discriminant equation and threshold value, respectively, to robustly separate A. besseyi and A. fujianensis, especially when Cq $ 35 are observed for an unknown sample of DNA.…”