Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces

Abstract

Background: Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces.

Methods: To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods.

Results: The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10^-1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections.

Conclusions: The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Keywords: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a); Diagnostic method; Fecal sample; Opisthorchiasis; Opisthorchis viverrini; Recombinase polymerase amplification.

Fig. 1

Map of Thailand showing the location of the field sites. Specific field collection sites are marked in the enlargements

Fig. 2

A Fluorescence values of the reactions containing Opisthorchis viverrini DNA and other helminth DNA were detected using a CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc.). Results are displayed as mean ± SD of three independent experiments. *** P < 0.001, ****P < 0.0001. B The image shows exposure of finished reaction tubes (Ov-RPA–CRISPR/Cas12a) to UV light. Only O. viverrini exhibited fluorescence when exposed to UV light. Tubes 1–8: nontemplate control (NTC), Al (A. lumbricoides), Tt (T. trichiura), Na (N. americanus), Ht (H. taichui), Cs (C. sinensis), Fg (F. gigantica), and Ov (O. viverrini, positive control). All reaction tubes were visually inspected using an UV transilluminator and photographed using a phone camera. C The image shows agarose gel electrophoresis of the RPA products. Only the reaction containing O. viverrini DNA showed the target amplicon at 281 bp. Lane M: 1 kb ladder (GeneRuler 1 kb Plus DNA Ladder, Thermo Fisher Scientific Inc.). Lanes 1–8: NTC, A. lumbricoides, T. trichiura, N. americanus, H. taichui, C. sinensis, F. gigantica, and O. viverrini. One of the three replicates was subjected to 1% gel electrophoresis (0.5× TBE, 100 V, ~ 35 min) and UV light assessment

Fig. 3

A Fluorescence values of tenfold serial dilutions of Opisthorchis viverrini DNA in Milli-Q sterile water. Results are displayed as mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001. B The image shows the exposure of finished reaction tubes (Ov-RPA–CRISPR/Cas12a) to UV light. Tubes 1–8: NTC, O. viverrini DNA at concentrations of 10, 1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ ng after dilution with Milli-Q sterile water. All reaction tubes were visually inspected using an UV transilluminator and photographed using a phone camera. C The image shows agarose gel electrophoresis results of purified RPA products obtained from finished reactions. Lane M: 1 kb ladder. Lanes 1–8: NTC, O. viverrini DNA at concentrations of 10, 1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ ng. The target amplicons (281 bp) were amplified at O. viverrini DNA concentrations of 10, 1, and 10⁻¹ ng. One of the three replicates was subjected to gel electrophoresis and UV light assessment. D Fluorescence values of NTC, NC (O. viverrini-negative human copro-DNA), and O. viverrini DNA at concentrations of 10, 1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ ng spiked in 100 ng of NC. E Tubes 1–9: NTC, NC, O. viverrini DNA at concentrations of 10, 1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ ng spiked in 100 ng of NC. F Lane M: 1 kb ladder. Lanes 1–9: NTC, NC, O. viverrini DNA at concentrations of 10, 1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ ng. Lane 10: 10⁻¹ ng of O. viverrini DNA (spiked in Milli-Q sterile water). The target amplicons (281 bp) were amplified at O. viverrini DNA concentrations of 10, 1, and 10⁻¹ ng (red arrowhead)

Fig. 4

A Representation of the results of finished reaction tubes (Ov-RPA–CRISPR/Cas12a) when exposed to UV light. On the basis of KK and/or FECT methods, tube NC contained O. viverrini-negative human copro-DNA, and tubes 1, 2, 5, 6, 7, 8, 10, 13, and 14 were positive for O. viverrini-like eggs. Tube 4 showed positive results for the coinfection of O. viverrini-like eggs and hookworms. Tubes 9 and 12 exhibited positivity for the coinfection of O. viverrini-like eggs and Taenia sp., respectively. Tubes 3 and 11 were positive for hookworms and S. stercoralis, respectively. Tube 15 represented a negative fecal sample, whereas tube PC served as a positive control with a plasmid containing the O. viverrini NAD1 gene. All reaction tubes were visually inspected using an UV transilluminator and photographed using a phone camera. B The image was recorded using the Gel Doc XR + Gel Documentation System. The tube’s label is based on image A. C The image shows agarose gel electrophoresis results of RPA products, with the red arrow indicating the target amplicon (281 bp). The lane labels align with images A and B. Additional details, including the results of KK and/or FECT methods, number of eggs per gram (NEPG), and Ov-RPA–CRISPR/Cas12a assay, are shown below the image. Lane M: 1 kb ladder. 1% gel electrophoresis (0.5× TBE, 100 V, ~ 35 min)

Fig. 5

Receiver operating characteristic (ROC) curve analysis of the comparison between Ov-RPA–CRISPR/Cas12a assay and the standard methods (KK and/or FECT) for detecting O. viverrini infection in field-collected fecal samples (n = 121)