We use cookies to understand how you use our site and to improve your experience. This includes personalizing content and advertising. To learn more, click here. By continuing to use our site, you accept our use of cookies. Cookie Policy.

Features Partner Sites Information LinkXpress
Sign In
Advertise with Us
INTEGRA BIOSCIENCES AG

Download Mobile App




Events

09 Apr 2024 - 12 Apr 2024
15 Apr 2024 - 17 Apr 2024
23 Apr 2024 - 26 Apr 2024

Rapid DNA Amplification Assay Detects Cryptosporidium in Stool Samples

By LabMedica International staff writers
Posted on 19 Feb 2014
Print article
Image: Nomarski interference contrast photomicrographs of Cryptosporidium muris from the feces of an HIV-positive human. Scale bars = five micrometers (Photo courtesy of the CDC – [US] Centers for Disease Control and Prevention).
Image: Nomarski interference contrast photomicrographs of Cryptosporidium muris from the feces of an HIV-positive human. Scale bars = five micrometers (Photo courtesy of the CDC – [US] Centers for Disease Control and Prevention).
Image: Strips developed at Rice University to look for the diarrheal disease cryptosporidiosis show the difference between positive and negative test results. Control stripes on the right show both tests are valid, while the presence of a second stripe near the center of the top strip shows that parasites are present in the patient\'s stool (Photo courtesy of Rice University).
Image: Strips developed at Rice University to look for the diarrheal disease cryptosporidiosis show the difference between positive and negative test results. Control stripes on the right show both tests are valid, while the presence of a second stripe near the center of the top strip shows that parasites are present in the patient\'s stool (Photo courtesy of Rice University).
A newly developed RPA (Recombinase Polymerase Amplification) lateral flow assay rapidly and accurately detected the parasite Cryptosporidium in stool samples.

Cryptosporidiosis, one of the most common waterborne diseases found worldwide, is a parasitic disease caused by the protozoan parasite Cryptosporidium. The parasite is transmitted by environmentally hardy microbial cysts (oocysts) that, once ingested, exist in the small intestine and result in an infection of intestinal epithelial tissue. The parasite disturbs the intestines and typically causes acute, self-limiting diarrhea. In immunocompromised individuals, such as AIDS patients, the symptoms are particularly severe and often fatal. Treatment is symptomatic, with fluid rehydration, electrolyte correction, and management of any pain.

Investigators at Rice University (Houston, TX, USA) have developed a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the RPA isothermal amplification technique to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 minutes; products are then detected visually on simple lateral flow strips.

Recombinase Polymerase Amplification (RPA) is a single tube, isothermal alternative to the Polymerase Chain Reaction (PCR) method for DNA application. Since it is isothermal, RPA reactions operate with simpler equipment than PCR, which requires a thermal cycler. The RPA process employs three core enzymes—a recombinase, a single-stranded DNA-binding protein (SSB), and strand-displacing polymerase. Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. SSB bind to displaced strands of DNA and prevent the primers from being displaced. Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA. By using two opposing primers, much like PCR, if the target sequence is indeed present, an exponential DNA amplification reaction is initiated. No other manipulation of the sample such as thermal or chemical melting is required to initiate amplification. At optimal temperatures (37–42 degrees Celsius), the reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 5 to 10 minutes,

An 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 stools of infected mice, and it correctly identified the presence or absence of the parasite in 27 out of 28 stool samples. Finally, the assay was tested using DNA extracted from the stool of infected patients, and it correctly identified the presence or absence of the parasite in 21 out of 21 stool samples.

The RPAC assay was integrated into a foldable, paper and plastic device that enabled DNA amplification with only the use of pipettes, pipette tips, and a heater. The method required little equipment, as the enzymes that amplify DNA worked best at or near body temperature. The performance of the integrated assay was comparable to or better than PCR, without requiring the use of thermal cycling equipment. Furthermore, RPA enzymes are stable in dried form and can be stored safely without refrigeration for up to a year.

“Diarrheal illness is a leading cause of global mortality and morbidity,” said senior author Dr. Rebecca Richards-Kortum, professor of bioengineering at Rice University. “Parasites such as Cryptosporidium are more common causes of prolonged diarrhea. Current laboratory tests are not sensitive, are time-consuming, and require days before results are available. A rapid, affordable, accurate point-of-care test could greatly enhance care for the underserved populations who are most affected by parasites that cause diarrheal illness.”

The study was published in the January 30, 2014, online edition of the journal Analytical Chemistry.

Rice University 

Platinum Member
COVID-19 Rapid Test
OSOM COVID-19 Antigen Rapid Test
HLX
Anti-Cyclic Citrullinated Peptide Test
GPP-100 Anti-CCP Kit
Gold Member
ADAMTS-13 Protease Activity Test
ATS-13 Activity Assay

Print article

Channels

Clinical Chemistry

view channel
Image: Reaching speeds up to 6,000 RPM, this centrifuge forms the basis for a new type of inexpensive, POC biomedical test (Photo courtesy of Duke University)

POC Biomedical Test Spins Water Droplet Using Sound Waves for Cancer Detection

Exosomes, tiny cellular bioparticles carrying a specific set of proteins, lipids, and genetic materials, play a crucial role in cell communication and hold promise for non-invasive diagnostics.... Read more

Molecular Diagnostics

view channel
Image: The cobas Malaria test is the first FDA-approved molecular test to screen U.S. blood donors for malaria (Photo courtesy of Roche)

First FDA-Approved Molecular Test to Screen Blood Donors for Malaria Could Improve Patient Safety

Malaria, a serious illness that often leads to death, is spread by a specific mosquito species that infect humans with a parasite. Other transmission modes include blood transfusions, organ transplants,... Read more

Hematology

view channel
Image: The low-cost portable device rapidly identifies chemotherapy patients at risk of sepsis (Photo courtesy of 52North Health)

POC Finger-Prick Blood Test Determines Risk of Neutropenic Sepsis in Patients Undergoing Chemotherapy

Neutropenia, a decrease in neutrophils (a type of white blood cell crucial for fighting infections), is a frequent side effect of certain cancer treatments. This condition elevates the risk of infections,... Read more

Pathology

view channel
Image: The medical office procedure detects the key biomarker in Parkinson’s and related neurodegenerative diseases (Photo courtesy of BIDMC)

Simple Skin Biopsy Test Detects Parkinson’s and Related Neurodegenerative Diseases

Parkinson's disease and a group of related neurodegenerative disorders known as synucleinopathies impact millions globally. These conditions, including Parkinson’s disease (PD), dementia with Lewy bodies... Read more
Copyright © 2000-2024 Globetech Media. All rights reserved.