Table 1.
The primers and probes used in the two multiplex reactions.
Fig 1.
Workflow of the laboratory procedures.
The three-staged approach for detection of soil-transmitted helminths in potassium dichromate preserved stool. Images used under license from Shutterstock.com.
Fig 2.
Effects of storing washed stools at -30°C on DNA.
SYBR green qPCR for the vertebrate mitochondrial gene was used to determine DNA integrity following “immediate” extraction from stools (stored 4°C for ≤4 weeks, ●) versus stools washed and stored at -30°C (□) for different lengths of time: 1 w (n = 6), 5 w (n = 4) and 15 w (n = 2). Mean Cqs and standard deviations are shown for duplicate reactions.
Fig 3.
Comparison of two homogenisers for DNA extraction.
The FastPrep-24™ 5G (FP) and Mini-Beadbeater-24 (BB) were used in conjunction with the Isolate II Fecal Kit to detect S. stercoralis (Ss) larvae and A. lumbricoides (Al) extracted from human stool by qPCR. Stool was equally divided into 8 aliquots and extraction was performed on either the FP or BB. Four different cycling times were tested using each homogeniser and the STH1 qPCR was performed in triplicate to ascertain mean Cqs and standard deviations. *p<0.05; **p<0.005 using paired t tests.
Fig 4.
The STH1 and STH2 qPCRs performed as both singleplex and multiplex reactions.
Synthetic gBlocks® (IDT) mimicking sequences from each of the six parasites were diluted 10-fold to generate 7-point standard curves. Mean Cq values and standard deviations from duplicate wells are shown.
Table 2.
Analytical performance of the two multiplex assays.
Table 3.
Validation of the STH1 and STH2 qPCRs.
Fig 5.
Trichuris suis spiked stool experiment.
Healthy stool was spiked with Trichuris suis eggs ranging from 0 to 640 EPG of stool and divided equally into four groups (two replicates per kit). DNA was extracted using either the Bioline fecal kit (per our protocol) or the Qiagen Powerfecal Kit incorporating 0.7 mm garnet beads. Each symbol is the mean and standard deviation from duplicate qPCR reactions.