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

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1 Accepted Manuscript Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry Kosala G. Weerakoon, Catherine A. Gordon, Geoffrey N. Gobert, Pengfei Cai, Donald P. McManus PII: S (16)30046-X DOI: doi: /j.mimet Reference: MIMET 4861 To appear in: Journal of Microbiological Methods Received date: 25 January 2016 Revised date: 3 March 2016 Accepted date: 21 March 2016 Please cite this article as: Weerakoon, Kosala G., Gordon, Catherine A., Gobert, Geoffrey N., Cai, Pengfei, McManus, Donald P., Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry, Journal of Microbiological Methods (2016), doi: /j.mimet This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 REVISED Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: a duplex approach with DNA binding dye chemistry Kosala G. Weerakoon a,b,c*, Catherine A. Gordon a, Geoffrey N. Gobert a1, Pengfei Cai a, Donald P. McManus a a Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia. b School of Public Health, University of Queensland, Brisbane, Australia. c Department of Parasitology, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka. *Corresponding author: Kosala.Weerakoon@qimrberghofer.edu.au (K.G. Weerakoon) 1 Present address: Queen s University Belfast, School of Biological Sciences, Belfast, United Kingdom. 1

3 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. Keywords Schistosomiasis; Schistosoma japonicum; Droplet digital PCR; Diagnosis 2

4 1. Introduction Schistosomiasis is a neglected tropical disease (NTD) which has a significant clinical impact on human mortality and morbidity (Hotez et al., 2014; Murray et al., 2012). The number of disability adjusted life years (DALYs) lost due to schistosomiasis in 2013 was reported as 3.1 million, representing ~14% of all DALY s lost due to all NTDs (Murray et al., 2015). It is estimated that schistosomiasis causes ~41,000 deaths annually and ~700 million people are at risk of infection globally (WHO, 2010, 2008). There are three main species of schistosomes infecting humans, Schistosoma mansoni, endemic to Africa, the Middle East, and South America, S. haematobium endemic to Africa, and S. japonicum, endemic to regions of South East Asia specifically in China, the Philippines and Indonesia. Schistosomiasis japonica, caused by S. japonicum, is a zoonosis with water buffaloes and other bovines being major reservoirs for human infection in both China and the Philippines, significantly complicating the control and elimination of the disease (Gordon et al., 2015; Gordon et al., 2012; Gray et al., 2009; Guo et al., 2006). The World Health Organisation (WHO) strategic plan for has determined that the goal of elimination of schistosomiasis, as a public health problem, is possible by 2025 (WHO, 2013). To achieve this outcome, there are a number of prevention and control programs currently underway. Accurate diagnostics are imperative to confirm the schistosomiasis elimination goals and to modulate treatment strategies at different transmission thresholds. Moreover, most schistosomiasis control programs are focussed on mass drug administration (MDA) or the targeted treatment of cases with the drug praziquantel. Hence, following regular praziquantel treatment regimens, disease prevalence and intensity of infection in endemic regions may be reduced, but elimination requires confirmation. Consequently, it is important to have available sensitive diagnostic tools that can detect low prevalence and low intensity infections, particularly within the framework of population surveillance and the monitoring of schistosomiasis control programs. Conventional parasitological diagnostic methods, such as the Kato-Katz (KK) thick smear examination or the miracidial hatching test (MHT), lack the requisite sensitivity in areas with low schistosome infection rates (Kongs et al., 2001; Spear et al., 2011; Yu et al., 2007). The majority of currently available diagnostic tests can only detect a patent schistosome infection, so the earlier point-of-care diagnosis of a schistosomiasis case, would be invaluable as it can guide the appropriate management of the disease (Gryseels et al., 2006; Liu et al., 3

5 2014). Moreover, currently, the diagnosis of schistosomiasis relies primarily on the direct microscopic detection of parasite ova, generally using the KK procedure, or by serology (Cai et al., 2014; WHO, 2013; Yu et al., 2007). While the KK is cheap and easy to implement, it can only detect patent infections and has relatively low sensitivity, particularly with light infections that occur in low disease transmission areas (Berhe et al., 2004; Kongs et al., 2001; Yu et al., 2007). In the case of S. japonicum, oviposition occurs 4-6 weeks post-infection and egg detection by microscopy can lack sensitivity due to variability in egg release and sampling related issues such as clumping of eggs and limited egg excretion in low intensity infections (Berhe et al., 2004; Kongs et al., 2001; Yu et al., 2007). Serological techniques have low specificity and high cross reactivity, and cannot differentiate between an active and a past infection (Doenhoff et al., 2004; Xie et al., 2014). Recent developments have shown that circulating antigen-based detection assays provide improved accuracy compared with antibody detection assays (Foo et al., 2015; Tchuem Tchuenté et al., 2012; VanDam et al., 2015). However, day-to-day fluctuations in assay results suggest the need for multiple sample testing to increase diagnostic sensitivity, particularly in low endemicity areas and low intensity infections (Legesse and Erko, 2007; Stothard et al., 2006; Tchuem Tchuenté et al., 2012). A more sensitive approach for improved schistosome diagnosis, including the capability to detect a pre-patent infection, is required. The detection of parasite cell free DNA (cfdna) in different clinical samples, is a recent valuable advance which provides significant benefits for the accurate diagnosis of a number of parasitic infections, including schistosomiasis (Enk et al., 2012; Kato-Hayashi et al., 2015, 2013). cfdna is comprised of fragments of extracellular DNA found mainly in the blood circulation, but it can also be detected in other bodily fluids such as saliva and urine. Of the range of DNA detection methods developed to date, the droplet digital polymerase chain reaction (ddpcr) provides high-precision, absolute quantification of nucleic acid target sequences, and has been shown to be even more sensitive than qpcr particularly in pathogen detection, cancer and prenatal testing (Hudecova, 2015; Miotke et al., 2014; Strain et al., 2013; Sze et al., 2014). Here we describe the development of a novel ddpcr method, with improved detection sensitivity compared to other molecular assays. The assay utilises two specific and abundant target gene sequences in a duplex assay for the diagnosis of S. japonicum infection. 4

6 2. Materials and methods: 2.1. Ethics statement This study was conducted with the approval (approval numbers P288, P443) of the QIMR Berghofer Medical Research Institute Animal and Human Ethics committees, and was performed in accordance with the Australian code of practice for the care and use of animals for scientific purposes, 7th edition (2004) Parasites Chinese and Philippine populations of S. japonicum adult worms were collected by perfusion of female ARC Swiss mice infected percutaneously with cercariae of the respective strains. S. japonicum cercariae from China (Guichi County, Anhui) and the Philippines (Irosin, near Sorsogon, Southern Luzon) were shed from infected Oncomelania hupensis hupensis and Oncomelania hupensis quadrasi snails, respectively. Mechanical transformation of cercariae to schistosomules, and their subsequent in vitro cultivation was performed as described (Milligan and Jolly, 2011). Schistosome eggs were isolated from livers of infected mice by digestion with collagenase B (Dalton et al., 1997) DNA samples and processing S. japonicum genomic DNA was extracted from adult worms, schistosomules and eggs of Philippine S. japonicum (SjP) and Chinese S. japonicum (SjC) using the PrepEase DNA isolation kit (Affymetrix, Santa Clara, USA) following the guidelines of the manufacturer. Genomic DNA was isolated from ARC Swiss mouse stool samples using the QIAmp DNA stool mini kit (Qiagen, Hilden, Germany). DNA quantification and quality testing was done using a Nanodrop 1000 (Thermo Scientific, Waltham, USA) spectrophotometer Selection of target genes and primer design The specific and conserved mitochondrial NADH dehydrogenase 1 (nad1) and retrotransposon SjR2 genes were selected as target sequences, as these sequences are highly abundant in the S. japonicum genome (Lier et al., 2006; Schistosoma japonicum genome sequencing and functional analysis consortium, 2009). The primers employed for the PCR amplification of a 82 bp fragment of the nad1 mitochondrial gene (GenBank Accession No: NC002544) have been previously reported (Gordon et al., 2012; Lier et al., 2006): 5'- TGRTTTAGATGATTTGGGTGTGC-3' (Forward) and 5'- 5

7 AACCCCCACAGTCACTAGCATAA-3' (Reverse). A pair of new primers (20 bp in length) were designed to amplify a 191 bp fragment of the highly repetitive SjR2 gene (GenBank Accession No: AY ), using the online primer-design software Primer3 ( 5'-CAGTGAAGTTGTGAAGGCTA-3' (Forward) and 5'- TAAAGACCGGAATGGTTAGT-3' (Reverse). Amplicons of differing sizes were selected to allow visualisation of both products for a ddpcr duplex assay Verification of primer design Conventional PCR (cpcr) Initial testing and optimisation of the PCR assays for the amplification of the two gene targets was done using genomic DNA extracted separately from schistosomules, adult worms and eggs of SjP and SjC as template. No DNA template controls (negatives) were used in all the assays and additionally DNA from some other relevant species (see below) were tested to determine the assay specificity. Reaction mixtures were prepared containing µl of MyTaq DNA polymerase (Bioline, London, UK), 0.5 µl of each primer from an initial 10 µm solution (5 pmol) (Sigma-Aldrich, St. Louis, USA), 5 µl of MyTaq PCR Buffer mix (Bioline), 2 µl of template DNA and distilled H 2 O to a final volume of 25 µl. The following cycling conditions were used for conventional PCR (cpcr) with the SjR2 primers: initial denaturation at 95 C for 2 min, followed by 35 cycles of 95 C (15 seconds), annealing at 55 C (15 seconds) and extension at 72 C (15 seconds), and a final extension at 72 C for 2 min. With the nad1 primers the cycling conditions were identical except for the annealing temperature which was increased to 59 C for 20 seconds. The PCR products were analysed visually after electrophoresis through 2% (w/v) agarose gels stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, USA). To evaluate the sensitivity of the cpcr assays with each primer, 1:10 serial dilutions of the total genomic DNA from SjP and SjC worms were used as the DNA template in the PCR. The length of the amplicon, analysed by gel electrophoresis, was checked for the expected band size. The amplification product was sequenced, and the resulting sequence compared to sequences in Genbank with the Basic Local Alignment Search Tool (BLAST) considering the identity and Expect (E) value parameters. E value refers to the number of hits that would be expected by chance when searching a data base for a particular sequence and it generally 6

8 describes the random background noise. A lower E value denotes a higher significance of the match. Specificity was further tested by using genomic DNA from S. mansoni, Taenia solium, Fasciola hepatica, Hymenolepis diminuta and Echinococcus granulosus and uninfected mouse and uninfected human serum (QIMR Berghofer volunteer) as DNA template in conjunction with the SjR2 and nad1 primers Real time PCR (qpcr) Reaction mixtures with a total volume of 15 μl were prepared containing 7.5 μl QuantiNova SYBR Green (Qiagen), 0.5 µl of each primer from an initial 10 µm solution (5 pmol), 5.5 μl of distilled H 2 O and 1 μl of DNA template. The PCR cycling conditions were as follows: 3 min initialization and denaturation at 95 C, followed by 40 cycles of 10 seconds denaturation at 95 C and 20 seconds annealing and extension at 60 C. The qpcr was performed using a real time thermocycler (Corbett RotorGene 6000, Hilden, Germany). Verification of amplification and the absence of primer dimer formation were checked by analysing SjP and SjC worm genomic DNA by melt-curve analysis, which resulted in a single peak at the expected temperature Development and optimisation of duplex ddpcr assay with EvaGreen ddpcr assays for each primer set were optimised separately, and then optimised as a duplex. Reaction mixtures of 20 µl were made comprising of 1 x ddpcr EvaGreen master mix (Bio- Rad, Hercules, USA) and forward and reverse primers in appropriate combinations and concentrations (Post optimisation: for singleplex assays, 2 µl of primers (forward and reverse) from 2 µm solutions for both SjR2 and nad1; for the duplex assay, this consisted of 2 µl of 2 µm SjR2 primers and 2 µl of 1 µm nad1 primers. Thus the final concentrations of primers for the duplex assays were 100 nm and 50 nm for the SjR2 and nad1 primers, respectively. In all assays the mixture included 2 µl of template DNA and distilled H 2 O to a final reaction volume of 20 µl. The reaction mix was pipetted into the sample well of an 8 channel disposable droplet generator cartridge (BioRad). For each cartridge, 70 µl of droplet generation oil for EvaGreen (BioRad) was loaded into oil wells, covered with a rubber gasket and then placed into a droplet generator (Bio-Rad). The generation of droplets resulted in partitioning the 20 µl sample into ~20,000 nanolitre sized droplets and the partitioned nanotlitre droplets were transferred to a 96-Well twin.tec PCR plate (Eppendorf, Hamburg, 7

9 Germany). The twin.tec plate was then heat sealed with a pierceable foil heat seal (BioRad) using a PX1 PCR Plate Sealer (BioRad) and placed into a thermal cycler (c1000, BioRad). The post optimisation two-step ddpcr cycling conditions were as follows: an initial enzyme activation period of 5 min at 95 C, 40 cycles of denaturation at 95 C for 30 seconds, an annealing/extension temperature of 55.8 C for 1 min, followed by a single final dye stabilization step of 4 C for 5 min and then 95 C for 5 min. Following the PCR amplification, the plate was placed in a QX200 Droplet Reader (BioRad) for analysis. Droplets from each well were automatically aspirated and sent through a two colour fluorescent detector to identify the fluorescent amplitude of each well. Droplet analysis was performed using Quanta-soft (BioRad) software. Optimisation of the two step ddpcr assay focussed on the parameters of annealing/extension temperature, final primer concentrations and the input template DNA concentration. An initial temperature gradient for the annealing/extension step of C was performed followed by a C temperature gradient to determine the optimum temperature. In optimizing the primer concentration combinations, initially nm concentrations of both the SjR2 and nad1 primers in a 1:1 ratio were tested followed by further dilutions from nm. These primer dilutions were further tested at 2:1 and 1:2 ratios also. To detect the optimum concentration of input template DNA, a dilution series of DNA (150, 100, 75, 50 and 25 ng/µl), extracted from the stool of an experimentally infected mouse, was tested in the assay. SjR2 and nad1 amplicon measurements obtained with the duplex assay were compared with those of the singleplex assays and the statistical significance was determined by Student s t-test using GraphPad Prism version 6.05 (GraphPad software, Inc., CA, USA). 3. Results 3.1. Detection of target sequences by cpcr The minimum detection levels with the cpcr were determined as 0.1 pg with both the SjR2 and nad1 primer sets (Figure 1). Both targets were amplified and detected in DNA from schistosomules, adult worms and liver eggs in the duplex cpcr assay (Figure 2). The optimum PCR cycling conditions for the SjR2 and nad1 primers differed. However, the nad1 primers amplified the target products under the same conditions as used with the SjR2 primers, but with a relatively low yield, as shown by the low gel intensity band (Figure 2). No 8

10 products were amplified when 50 ng/µl genomic DNA from S. mansoni, T. solium, F. hepatica, H. diminuta, E. granulosus, uninfected mouse and uninfected human serum were used as DNA template. Comparison of the target sequence with sequences in Genbank with the BLAST algorithm exhibited 100% identity (E value = 5e -94 ) for S. japonicum Duplex ddpcr assay with EvaGreen ddpcr assays for the detection of the two targets were initially designed as singleplex assays and were then further adapted into a duplex assay to detect both amplicons concurrently (Figure 3). As the SjR2 product (191 bp) is longer than the nad1 fragment (82 bp), droplets with SjR2 amplicons showed higher fluorescence amplitude. Therefore, the two amplicons could be identified as two separate clusters in the same assay (Figure 3). Quantification of the two target genes was performed with a 2D droplet plot, using 2D clustering tools to assign separate droplet populations. The resulting SjR2 and nad1 amplicon concentration measurements obtained with the duplex assay were similar to those of the singleplex assays (Figure 4). The overall strategy for the optimisation of the duplex ddpcr assay with final optimum conditions is presented in Figure Optimum temperature for the ddpcr assay Using the initial conditions, positive and negative clusters were identified, with rain (droplets with intermediate fluorescence and thus non-specific or indeterminate amplicon) between the clusters (Figure 6). The temperature gradient ddpcrs showed that at high (>61 C) temperatures, amplification was low and with low temperatures (<54 C) the assay showed increasing nonspecific amplifications with high amounts of rain droplets affecting cluster separation (Figures 5 and 7). A temperature of 55.8 C was selected as the optimal annealing/extension temperature, since it resulted in the best balance of high yield and cluster differentiation in the duplex assay Optimum primer concentrations Of the different primer concentrations tested, higher concentrations of the SjR2 and nad1 primers at a 1:1 ratio showed a high amount of rain, while lower dilutions showed relatively low amounts of rain. Multiple primer concentration ratios were tested and it was observed that a higher primer concentration (100 nm) for SjR2 and lower primer concentration (50 nm) for nad1 were found to be optimal, resulting in a more precise outcome (Figures 5 and 7). 9

11 3.5. Optimum template DNA concentration A dilution series of DNA extracted from the stool of an infected mouse was used to determine the optimum concentration of template DNA for the assay. At high DNA concentrations, the total number of droplets generated was low, and had a higher incidence of rain. A final concentration of 5 ng/µl in a 20 µl reaction mix (using 2 µl of 50 ng/µl DNA) was confirmed as optimal (Figures 5 and 7) Minimum detection level The minimum detection level of the assay was determined using a serial dilution of S. japonicum worm genomic DNA. The assay was able to detect as little as 0.05 fg of template DNA, which is considerably less than the total DNA content of a pair of adult worms (about 3000 ng) or a single egg (about 50 pg). 4. Discussion DNA-based detection methods provide a viable alternative to some of the commonly used tests for the diagnosis of schistosomiasis japonica (Gordon et al., 2015; Gordon et al., 2011; Xu et al., 2010). However, it is important to consider the value of testing clinical samples using different bodily fluids with DNA detection-based assays. For example, PCR assays performed on DNA extracted from faecal samples use a similar amount of faecal material (200 mg) as the optimal triplicate KK slides (150 mg total, 50 mg per slide) currently used (Gordon et al., 2015). The main sources of Schistosoma DNA in a faecal sample are the eggs, and the chance of finding an egg in a positive stool sample is similar for the KK and by PCR, although the latter is considerably more sensitive, and is not compromised by a microscopist failing to identify and therefore missing an egg. A conventional copro-pcr assay may not detect an early pre-patent infection before oviposition at ~4-6 weeks, post cercarial infection (Ross et al., 2002). However, there are reports showing high sensitivities and early detection profiles (before patency) in animal models when examining faecal DNA, indicating the likely presence of parasite cell free (cf) DNA (Fernández-Soto et al., 2014; Gordon et al., 2015). Schistosome cfdna has been detected in different clinical samples, such as urine, blood and saliva and represents a valuable biomarker for detection of an early infection (Enk et al., 2012; Kato-Hayashi et al., 2015, 2013). Schistosomes are multi-cellular parasites that contain multiple DNA copies of target genes. Life stages (schistosomules, adults, eggs) are in direct 10

12 contact with the circulatory system, so it is likely that parasite cells or parasite tissue fragments are constantly shed into the circulation so that DNA of parasite origin can be found in the blood. However it is likely that a very low amount of parasite cfdna would be present in a blood sample, due to the dilution of the cfdna in a large volume of plasma. Therefore the concentrations of schistosome cfdna in other body fluids such as urine (with the exception of S. haematobium which parasitises the bladder) and saliva, where direct contact with the parasite stages does not occur and the exact mechanisms of their release is yet unclear, cfdna will be present in low amounts (Weerakoon and McManus, 2016). Hence, particularly in low intensity infections, cfdna detection requires a highly sensitive diagnostic approach, using more technically advanced, novel DNA based methods, such as ddpcr (Hudecova, 2015; Miotke et al., 2014; Strain et al., 2013; Sze et al., 2014). When compared with cpcr and qpcr assays, ddpcr has higher diagnostic accuracy, and is able to provide absolute quantification, both features being beneficial in the support of disease control and elimination programs. These characteristics are particularly important in low schistosomiasis transmission areas with low prevalence/intensity infections common in areas in China where an extensive control program has been ongoing for many years (Minggang and Zheng, 1999; Yuan et al., 2002). Here we describe a duplex ddpcr assay for the detection of S. japonicum infection through the amplification of a retrotransposon (SjR2) and a mitochondrial gene (nad1) sequence. By utilising these highly abundant targets in a duplex assay, we have increased the diagnostic sensitivity of the test. This approach increases the chance of accurately detecting low amounts of parasite DNA within more abundant host DNA in stool or body fluids sampled. The assay represents an ideal tool for the detection of low levels of parasite DNA (including cfdna) in different clinical samples. The assay detected target sequences in different sources of Schistosoma DNA isolated from adult worms, schistosomules and eggs; it is highly sensitive and can detect very low amounts of parasite DNA (Lier et al., 2006; Xia et al., 2009; Xu et al., 2010). The specificity of the assay is high, with no cross reactivity found when tested using DNA from other helminth parasites as template, nor was there any crossreactivity with host DNA. In addition to target detection, the assay has the potential for directly quantifying the amount of target sequences in a sample. In future this could be applied in the assessment of infection intensity, following validation in vivo and using appropriate human clinical and animal samples. 11

13 The development of multiplex qpcr assays requires custom-made fluorescent probes which increase the complexity and costs involved. For cpcr and ddpcr, multiplexing can be achieved without fluorescent probes by designing primer sets which produce different amplicon lengths. With cpcr these amplicon sizes are distinguished by running on an agar gel which separates the amplicons based on size. In this way many different amplicons can be used; however cpcr lacks sensitivity when compared with qpcr- or ddpcr-based tests. Fluorescent probes can be used in ddpcr but duplexing is also possible with the use of EvaGreen dsdna binding dye. For ddpcr the final fluorescence signal amplitude is dependent on amplicon length. When two amplicons of different sizes are generated, these are shown as separate clusters with distinct amplitudes based on the fluorescent intensity. Hence this phenomenon can be used to design sensitive multiplex ddpcr assays without the added costs associated with probe-based assays. In the assay described here, the SjR2 amplicon (191 bp) is larger than the nad1 amplicon (82 bp), therefore droplets with SjR2 amplicons have a higher fluorescence amplitude than nad1 and the amplicons appear as two separate clusters in 1D and 2D fluorescence plots (Figure 4). Classification of positive and negative droplets in ddpcr needs a cut-off to facilitate the final analysis of results that are positive. However, not all droplets emit a fluorescent signal that can be classified readily as either positive or negative and hence it is problematical to determine the exact margins of the two clusters. These droplets in the indeterminate region between positive and negative clusters are generally known as rain. For a balanced analysis of these droplets, the standard QuantaSoft software provided with the Bio-Rad ddpcr system classifies droplets by first determining a fluorescence threshold and subsequently, all droplets with a fluorescence value greater than this threshold are considered positive. To calculate this threshold, every droplet is allocated to either a positive or negative cluster, and a proprietary method, based on poisson and binomial statistical algorithms, is applied to the data to define the fluorescence threshold. This automatic threshold can be manually adjusted for more or less stringent threshold limits. The differentiation and analysis of the two positive clusters in a duplex assay is done with 2D plots which provide apparent cluster identity and separation (Figure 3). In the presence of droplets between the two positive clusters, these will be separated across the median (Bio-Rad Laboratories Inc., 2014; Miotke et al., 2014). In addition to the two positive clusters (nad1 and SJR2 positive droplets), there is a possibility of having a third cluster of droplets containing double positives where the droplets can contain both target amplicons, resulting in a third, higher fluorescence amplitude (McDermott 12

14 et al., 2013). However in the assay described here, double positive clusters were not identified, possibly due to the low amount of target DNA concentration used which likely led to a much lower chance of two or more template DNA fragments being accommodated in a single droplet. By varying primer concentrations and annealing/ extension temperatures, we were able to determine the best conditions for high amplification of target sequences while providing good cluster separation. It is important to accommodate the highest possible amount of DNA in the reaction to achieve a maximum level of detection. This is particularly true for detecting low intensity S. japonicum infections where a very small amount of target parasite DNA is present in clinical samples. However, higher concentrations of template DNA affected the ddpcr reaction, reducing the yield and efficiency, and decreasing cluster separation. We noted that with higher amounts of template DNA, both droplet generation and amplification were impaired. The effect on droplet production may be due to chemical reactions at the oil water interface which are known to occur with higher DNA concentrations (Strain et al., 2013). Impaired droplet generation in turn affects the ddpcr reaction. Having high concentration of template DNA can also cause PCR inhibition. Moreover, with higher template DNA concentration the limited amplifications would not be clearly detected with the higher fluorescence levels from excessive background DNA. Negative samples can occasionally present one or two droplets in the positive amplitude region, which in turn affect the detection limits. In such situations, for improved clarity in assays, a positive assay result was defined as the presence of more positive droplets than the maximum number of positive droplets present in negative controls. These effects can be minimised with a probe based assay, since fluorescent emission is more specific to amplification process; however this increases the cost of the assay significantly. While ddpcr is the most sensitive diagnostic tool available, like many other molecular-based assays, it is expensive. Establishment costs for the required equipment, reagents and the technical expertise are limitations, particularly if considered as a routine diagnostic tool in poor resourced endemic communities (Gordon et al., 2011). However, the assays could play an important future role as a research tool in monitoring integrated control and elimination programs for schistosomiasis and other neglected parasitic infections. In summary, the novel dsdna binding dye based duplex ddpcr assay we have developed is sensitive and specific for the detection of S. japonicum infection and would be an ideal tool 13

15 for the sensitive detection of low amount of cfdna in the host circulation and other bodily fluids. The assay needs. In the future, after further validation in an animal model and with human clinical samples, this assay could be an invaluable asset for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas to support current WHO schistosomiasis elimination targets (WHO, 2013). Acknowledgements We thank Mary Duke (QIMR Berghofer Medical Research Institute) for help with animal experiments and Anthony Beckhouse (Bio-Rad Laboratories, Pty Ltd) for his advice and support with QX200 droplet digital PCR system. The project received funding support from the National Health and Medical Research Council (NHMRC) of Australia (Grant numbers: ID613671, APP ; APP ). DPM is a NHMRC Senior Principal Research Fellow and Senior Scientist at QIMR Berghofer Medical Research Institute. Figures Figure 1: Conventional PCR amplification of SjP and SjC worm DNA in a dilution series with SjR2 and nad1 primers. L: 100 bp ladder, Lanes 1-8: adult worm genomic DNA, 1:10 dilution series starting from 50 ng/ µl, Lane 9: No DNA template control. Panel A: Assay with SjR2 primers on SjP worm genomic DNA, Panel B: Assay with nad1 primers on SjP worm genomic DNA, Panel C: Assay with SjR2 primers on SjC worm genomic DNA, Panel D: Assay with nad1 primers on SjP worm genomic DNA. Figure 2: Conventional PCR amplification of genomic DNA from different stages of SjP and SjC with SjR2 and nad1 primers. L: 100 bp ladder, Lane 1 SjP adult worm genomic DNA, Lane 2 SjC adult worm genomic DNA, Lane 3 SjP schistosomule DNA, Lane 4 SjC schistosomule DNA, Lane 5 SjP egg DNA, Lane 6 SjC egg DNA, Lane 7 No DNA template control. 14

16 Figure 3: Visual representation of multiplexed target detection using EvaGreen-based ddpcr 1.0 pg of SjP worm genomic DNA amplified with nad1 and SjR2 primers: Panel A - 1D droplet plot: Negative droplets in the lowest cluster, positive SjR2 (191 bp) droplets in the highest cluster and the positive nad1 (82 bp) cluster in the middle cluster are shown. The pink line indicates the manually set fluorescence amplitude level; Panel B - 2D droplet plot: Negative droplets in the lowest cluster, positive SjR2 droplets in the highest cluster and the positive nad1 cluster in the middle cluster are shown. (Event number: number of droplets in the reaction, Pos: number of positive droplets, Neg: number of negative droplets, Amplitude: Fluorescent intensity from each droplet captured by the droplet reader). Figure 4: Comparison of singleplex and duplex ddpcr assays. Concentration measurements of the SjR2 and nad1 targets from singleplex and duplex reactions, with 1.0 pg of SjP worm genomic DNA analysed in three biological replicates. No significant differences (p value > 0.05) were recorded between the singleplex and duplex assays for the two target sequences. Error bars represent standard error of mean with n=3. Figure 5: Summary of main optimisation steps in the duplex ddpcr assay. Figure 6: Rain droplets in the duplex ddpcr assay. Two samples of SjP worm genomic DNA (A01 left and B01 right) were amplified with nad1 and SjR2 primers. Negative droplets in the lowest cluster (Grey colour), positive SjR2 (191 bp amplicon) droplets in the highest cluster and the positive nad1 (82 bp) cluster in the middle (both Blue colour) are shown. The positive and negative droplets, classified by the QuantaSoft automatic analysis of individual wells, are shown in blue and grey, respectively. The grey shaded area represents the zone containing rain droplets - droplets with intermediate fluorescence between the clusters. The automatic threshold set by QuantaSoft software can be manually adjusted for more or less stringent threshold limits. The differentiation and analysis of the two positive clusters in a duplex assay is done with 2D plots which provide apparent cluster identity and separation. In the presence of droplets between the two positive clusters, these will be separated across the median. (Event number: number of droplets in the reaction, Pos: number of positive droplets, 15

17 Neg: number of negative droplets, Amplitude: DNA binding dye fluorescent intensity from each droplet captured by the droplet reader). Figure 7: Optimisation of temperature, primer concentrations and input template DNA concentrations. Panel A- Temperature gradient ddpcr: A1 - Temperature from 65 o C 55 o C from left to right. No amplification of both targets and some nonspecific amplifications at higher temperatures. A2 - Temperature from 56 o C - 51 o C from left to right. High amount of nonspecific amplifications with more evident rain at lower temperatures. Panel B - Effects of different primer concentrations. Columns 1-3: assays with SjR2 final primer concentration in the reaction mix as 100, 75 and 50 nm with a constant nad1 primer concentration of 50 nm. Best amplifications and cluster separation at 100 nm: 50 nm concentration combination. Limited amplification of SjR2 with the primer concentration of 50 nm, resulting in a lower fluorescence amplitude affecting the cluster separation. Panel C: Effects of different template DNA concentrations. Comparison of different thresholds on serial dilutions of target DNA done with S. japonicum infected mouse faecal DNA. Columns 1-5 assays with 2 µl of template DNA at the concentration range, 150, 100, 75, 50 and 25 ng/µl. Best amplifications and cluster separation at 50 ng/µl concentration. Negative droplets in the lower most cluster, positive SjR2 (191 bp amplicon) droplets in the upper most cluster and the positive nad1 (82 bp) droplets in the middle cluster are shown. The pink line indicates the manually set fluorescence amplitude level. The positive and negative droplets, classified by the QuantaSoft automatic analysis of individual wells, are shown in blue and grey, respectively. (Event number: number of droplets in the reaction, Pos: number of positive droplets, Neg: number of negative droplets, Amplitude: Fluorescent intensity from each droplet captured by the droplet reader). 16

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