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

Crannell ZA, Castellanos-Gonzalez A, Irani A, Rohrman B, White AC, Richards-Kortum R - Anal. Chem. (2014)

Bottom Line: Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance.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.

View Article: PubMed Central - PubMed

Affiliation: Rice University , 6500 Main Street, Houston, Texas 77251, United States.

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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Related in: MedlinePlus

Gel detectionof RPA products. Amplified products were detectedusing gel electrophoresis stained with ethidium bromide. Using DNAextracted from oocysts spiked into PBS, RPA products from as few as103 oocysts/mL PBS (A) are visible on the gel. Using DNAextracted from oocysts spiked into uninfected stool samples from healthyvolunteers, RPA products from as few as 104 oocysts/mLstool (B) are visible on the gel.
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fig1: Gel detectionof RPA products. Amplified products were detectedusing gel electrophoresis stained with ethidium bromide. Using DNAextracted from oocysts spiked into PBS, RPA products from as few as103 oocysts/mL PBS (A) are visible on the gel. Using DNAextracted from oocysts spiked into uninfected stool samples from healthyvolunteers, RPA products from as few as 104 oocysts/mLstool (B) are visible on the gel.

Mentions: Using total nucleic acids extracted from PBS containingoocysts, amplified products from as few as 103 oocysts/mLPBS were detectable via gel electrophoresis (Figure 1A). When using nucleic acids extracted from stool, amplifiedproducts from as few as 104 oocysts/mL stool are detectablevia gel electrophoresis (Figure 1B).


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

Crannell ZA, Castellanos-Gonzalez A, Irani A, Rohrman B, White AC, Richards-Kortum R - Anal. Chem. (2014)

Gel detectionof RPA products. Amplified products were detectedusing gel electrophoresis stained with ethidium bromide. Using DNAextracted from oocysts spiked into PBS, RPA products from as few as103 oocysts/mL PBS (A) are visible on the gel. Using DNAextracted from oocysts spiked into uninfected stool samples from healthyvolunteers, RPA products from as few as 104 oocysts/mLstool (B) are visible on the gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3958140&req=5

fig1: Gel detectionof RPA products. Amplified products were detectedusing gel electrophoresis stained with ethidium bromide. Using DNAextracted from oocysts spiked into PBS, RPA products from as few as103 oocysts/mL PBS (A) are visible on the gel. Using DNAextracted from oocysts spiked into uninfected stool samples from healthyvolunteers, RPA products from as few as 104 oocysts/mLstool (B) are visible on the gel.
Mentions: Using total nucleic acids extracted from PBS containingoocysts, amplified products from as few as 103 oocysts/mLPBS were detectable via gel electrophoresis (Figure 1A). When using nucleic acids extracted from stool, amplifiedproducts from as few as 104 oocysts/mL stool are detectablevia gel electrophoresis (Figure 1B).

Bottom Line: Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance.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.

View Article: PubMed Central - PubMed

Affiliation: Rice University , 6500 Main Street, Houston, Texas 77251, United States.

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.

Show MeSH
Related in: MedlinePlus