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. 2014 Mar 4;86(5):2565-71.
doi: 10.1021/ac403750z. Epub 2014 Feb 12.

Nucleic acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens

Affiliations

Nucleic acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens

Zachary Austin Crannell et al. Anal Chem. .

Abstract

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens.

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Figures

Figure 1
Figure 1
Gel detection of RPA products. Amplified products were detected using gel electrophoresis stained with ethidium bromide. Using DNA extracted from oocysts spiked into PBS, RPA products from as few as 103 oocysts/mL PBS (A) are visible on the gel. Using DNA extracted from oocysts spiked into uninfected stool samples from healthy volunteers, RPA products from as few as 104 oocysts/mL stool (B) are visible on the gel.
Figure 2
Figure 2
Lateral flow detection of RPA product. Dual labeled amplicons can be detected visually using lateral flow strips (A). Anti-FITC conjugated gold nanoparticles dried in the sample pad bind to the FITC label on RPA amplicons. Gold nanoparticles wick down the strip where amplicon bound nanoparticles are captured at the streptavidin detection line and those nanoparticles not bound to amplicons are captured at the positive control line. RPA products from DNA extracted from as few as 102 oocysts/mL PBS (B) can be detected visually. RPA products from DNA extracted from as few as 102 oocysts/mL stool (C) can be detected visually.
Figure 3
Figure 3
Testing the RPAC assay for specificity. RPA products detected using lateral flow RPAC assay yield visually positive results only when tested using DNA extracted from PBS spiked with Cryptosporidium; results are visually negative for all other organisms tested.
Figure 4
Figure 4
RPA testing using a paper and plastic foldable device. (A) The reagents are first added to their respective pads, (B) the wick is then dipped into the tube containing DNA extracted from the stool specimen, (C) and the device is folded to initiate and perform the RPA reaction.

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