Compared with conventional PCR assay, qPCR assay greatly improves the detection efficiency of predation

Abstract

Studies of predation can contribute greatly to understanding predator–prey relationships and can also provide integral knowledge concerning food webs and multi‐trophic level interactions. Both conventional polymerase chain reaction (cPCR) and quantitative PCR (qPCR) have been employed to detect predation in the field because of their sensitivity and reproducibility. However, to date, few studies have been used to comprehensively demonstrate which method is more sensitive and reproducible in studies of predation. We used a Drosophila melanogaster‐ specific DNA fragment (99 bp) to construct a tenfold gradient dilution of standards. Additionally, we obtained DNA samples from Pardosa pseudoannulata individuals that fed on D. melanogaster at various time since feeding. Finally, we compared the sensitivity and reproducibility between cPCR and qPCR assays for detecting DNA samples from feeding trials and standards. The results showed that the cPCR and qPCR assays could detect as few as 1.62 × 10 3 and 1.62 × 10 1 copies of the target DNA fragment, respectively. The cPCR assay could detect as few as 48 hr post‐feeding of the target DNA fragment. But the qPCR assay showed that all spiders were positive after consuming prey at various time intervals (0, 24, 48, 72, and 96 hr). A smaller proportion of the technical replicates were positive using cPCR, and some bands on the agarose gel were absent or gray, while some were white and bright for the same DNA samples after amplification by cPCR. By contrast, a larger proportion of the technical replicates were positive using qPCR and the coefficients of variation of the Ct value for the three technical replicates of each DNA sample were less than 5%. These data showed that qPCR was more sensitive and highly reproducible in detecting such degraded DNA from predator's gut. The present study provides an example of the use of cPCR and qPCR to detect the target DNA fragment of prey remains in predator's gut.

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces

“…In line with previous findings of 27 , 54 , 55 , qPCR had a higher sensitivity than cPCR. It proved to be a more reliable and robust assay to detect T. pityocampa in bat faecal samples for two main reasons.…”

A comparative analysis of spider prey spectra analyzed through the next‐generation sequencing of individual and mixed DNA samples

“…The predation relationship between spiders and prey is determined by detecting specific DNA fragments of prey remains in the spider's gut. Conventional polymerase chain reaction (cPCR), quantitative PCR (qPCR), and next-generation sequencing (NGS) technology have been used for molecular gut content analysis (Macías-Hernández et al, 2018;Yang, Liu, et al, 2020). Among them, cPCR and qPCR are suitable for studying predation on a single or a few target prey based on prey-specific primers (Yang, Liu, et al, 2020;Yang, Liu, Yuan, Zhang, Peng, et al, 2017).…”

“…Conventional polymerase chain reaction (cPCR), quantitative PCR (qPCR), and next-generation sequencing (NGS) technology have been used for molecular gut content analysis (Macías-Hernández et al, 2018;Yang, Liu, et al, 2020). Among them, cPCR and qPCR are suitable for studying predation on a single or a few target prey based on prey-specific primers (Yang, Liu, et al, 2020;Yang, Liu, Yuan, Zhang, Peng, et al, 2017). NGS is suitable for analyzing the prey composition of generalist predators (e.g., spiders) based on general primers for potential prey (Zhong et al, 2019).…”

Leishmania V. braziliensis infection in asymptomatic domestic animals within an endemic region in the Northeast of Brazil

“…When compared to qPCR, conventional PCR on leukocytes from C. familiaris had a positivity percentage that was almost 70% smaller, with statistical significance. This may be because qPCR is more sensitive to DNA detection than conventional PCR 23 , and to protocol variations among different systems. Several factors influence DNA detection in a molecular testing system, such as extraction methods, initial primers selection, clinical sample types, and infection period of each animal, and the latter is directly related to parasite load.…”