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Multiplex PCR Detection of Viral, Parasite and Bacterial Pathogens

Description:

Executive Summary
Infectious disease researchers at the University of Virginia have developed quantitative multiplex RT-PCR assays for the major viral, bacterial, protozoan and helminthes causes of infectious gastroenteritis.  

Background
Infectious gastroenteritis, or the “stomach flu,” results in acute diarrhea, vomiting and abdominal pain. In the United States, an estimated 211–375 million episodes of diarrheal illness occur each year, resulting in 73 million physician consultations, 1.8 million hospitalizations and 3,100 deaths. The economic costs of infectious diarrheal diseases are considerable, and each year an estimated $6 billion is spent in the U.S. on medical care and lost productivity.

When diagnosing infectious gastroenteritis, conventional diagnostic methods — such as culture, biochemical tests, PCR and ELISA — can be laborious, expensive and insensitive. Additionally, although infection with multiple pathogens is common, existing diagnostic methods are typically ordered and performed for individual pathogens, such that these individual assays may routinely miss the diversity of infecting organisms. 

About the Invention
The four multiplex RT-PCR panels listed below are capable of rapid, specific, sensitive and quantitative detection of the viral, bacterial and parasitic pathogens most commonly responsible for causing gasteroenteritis. 

Pathogen Sensitivity Specificity
Viruses    
Norovirus GI 100% 94%
Norovirus GII 95% 100%
Rotavirus 100% 97%
Astrovirus 100% 100%
Sapovirus 100% 88%
Adenovirus 100% 100%
Bacteria    
Aeromonas species 89% 87%
Campylobacter jejuni and coli 92% 92%
Salmonella species 96% 94%
Shigella species and EIEC 94% 93%
Vibrio cholera and parahaemolyticus 92% 96%
Yersinia species 100% 100%
Protozoa    
Giardia intestinalis 89% 97%
Cryptosporidium spp. 90% 100%
Entamoeba histolytica 86% 98%
Helminth    
Ancylostoma duodenale 100% 99%
Ascaris lumbricoides 100% 100%
Necator americanus 90% 95%
Strongyloides stercoralis 100% 97%

The limit of detection of the multiplex RT-PCR-Luminex assay correlates to 102–104 viral copies/g of stool; 103-105 bacterial cfu/g of stool; 103 Giardia cysts, 102 Cryptosporidium oocysts and 101 E. histolytica trophozoites/200mg stool; and 10−4, 10−6, 10−6 and 10−4 dilutions of confirmed samples, respectively, of Ancylostoma, Ascaris, Necator,and Strongyloides.

Advantages
This technology offers the following advantages over existing technologies:

  • Simultaneously identifies and quantifies infections by multiple pathogens
  • Allows assay directly from fecal samples
  • Offers comparable reliability to the gold standard assay
  • Completes testing in under six hours


Resources
For more information, please see the following inventor publications:

  • J Clin Virol. “Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis.” 2011 Apr;50(4):308-13. (Available online.)
  • Am J Trop Med Hyg. “High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites.” 2011 Feb;84(2):332-7. (Available online.)

 

Patent Information:
Category(s):
Diagnostics & Assays
For Information, Contact:
Stephanie Miller
Licensing Associate
UVA
stephanie@uvapf.org
Inventors:
Eric Houpt
Jie Liu
Keywords:
Diagnostics
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