Table 1.
Primers and probe sequences for the ITS2 of N. americanus and A. duodenale.
Fig 1.
The positions of the primers designed in this study.
Fig 2.
Results of primers and probe screening for ITS2 gene.
(a) Primer-probe interference experiments. (b) Optimization results of the primer combinations for detecting N. americanus eggs.
Fig 3.
Determination of the minimum ITS2 detection limit of the RPA assay using the optimal primer combination.
Curves 1- 7 shows the amplification fluorescence curves of N. americanus genomic DNA samples with pre-set concentrations of 100 pg/µL, 10 pg/µL, 1 pg/µL, 100 fg/µL, 10 fg/µL, 1 fg/µL, and 0.1 fg/µL, respectively. NTC: No template control.
Fig 4.
Cross reaction test of fluorescence RPA (a) Cross reaction test with C. sinensis, S. japonicum, F. hepatica, A. lumbricoides, E. vermicularis (Line 1-5: Genomic DNA of C. sinensis, S. japonicum, F. hepatica, A. lumbricoides and E. vermicularis; PC: Positive control; NC: Negative control; NTC: No template control) (b) Cross reaction test with A. duodenale (Line6: Genomic DNA of A. duodenale; PC: Positive control; NC: Negative control; NTC: No template control).
Fig 5.
Detection results of fecal samples containing varying quantities of N. americanus eggs.
(a) Detection results of fecal samples containing 1 egg by fluorescent RPA. (b) Detection results of fecal samples containing 3 eggs by fluorescent RPA.
Table 2.
Detection results of semi-nested PCR and fluorescence RPA assay for 20 samples each containing one egg of N. americanus.
Table 3.
Detection results of semi-nested PCR and fluorescence RPA assay for 20 samples each containing three egg of N. americanus.
Table 4.
Raw data of detection results of 41 samples using Kato-Katz, semi-nested PCR and fluorescent RPA.
Fig 6.
Detection results of N. americanus in 41 Human Fecal Samples via Fluorescent RPA method for Sensitivity and Specificity Assessment (Line 1-41: Human fecal samples; PC: Positive control; NC: Negative control; NTC: No template control).
Table 5.
Laboratory validation of the Kato-Katz for human fecal samples compared with fluorescent RPA and semi-nested PCR assays.
Fig 7.
Detection results of N. americanus in 287 human fecal samples collected from the field via Fluorescent RPA (Line 1-287: Human fecal samples from the field; PC: Positive control; NC: Negative control; NTC: No template control).
Fig 8.
Venn diagram of the positive results of the three assays.
Table 6.
Number of eggs in the 40 positive fecal samples detected by the Kato-Katz and the detection results of semi-nested PCR and fluorescent RPA on these samples.
Table 7.
Comparison of the Kato-Katz with semi-nested PCR for detecting human fecal samples.
Table 8.
Comparison of the Kato-Katz with fluorescent RPA for detecting human fecal samples.
Table 9.
Comparison of the fluorescent RPA with Semi-nested PCR for detecting human fecal.