Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Abstract

Schistosomiasis is a chronically debilitating helminth infection with a significant socioeconomic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostics procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR

“…Thus, results from this sample must be critically reviewed and droplet generation parameters should be included in the analysis of suspect samples. One possible explanation could be a high amount of DNA being present in these samples that was shown to affect droplet production [ 26 , 32 ]. Further, the sample DNA was stored over a longer period at −20 °C, which negatively influences DNA quality [ 9 ].…”

Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals

“…The current methods used in China for the diagnosis of S. japonicum infection in domestic animals are MHT and IHA. However, the diagnostic assays available to date are not ideal because the identification of miracidia in stools has a low sensitivity and antibody detection lacks specificity, which limits the determination of prevalence rates [14]. In this respect, PCR is a potential tool because of its high sensitivity and specificity in the diagnosis of schistosomiasis in humans [23].…”

“…Cell-free circulating DNA and some nucleic acid fragments were primarily found in host blood, saliva, semen and urine, and were used as targets for parasite detection [14–16]. Moreover, cell-free circulating DNA has been used as a marker for cancer and prenatal diagnosis [17].…”

“…Therefore, DNA extracted from host fluids could be used for early diagnosis [18]. Various PCR-based approaches, including real-time PCR, nested-PCR, and loop-mediated isothermal amplification, have been applied for detection of schistosome infection [14, 19, 20]. However, no previous studies have diagnosed S. japonicum infection in domestic animals by using PCR-based methods.…”

Persistence of Schistosoma japonicum DNA in a Kidney–Liver Transplant Recipient

“…The DNA was used in a digital droplet PCR (ddPCR) assay with primers which amplify the NADH1 dehydrogenase I (NADH1) mitochondrial gene of S. japonicum as described previously. [8][9][10][11][12][13] After amplification, reactions were read using a QX200 (Bio-Rad, Hercules, CA) droplet reader. Non-template controls using water in place of DNA, and positive controls using DNA from S. japonicum eggs isolated from the liver of an infected mouse were used in all PCR assays.…”