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Simple, Inexpensive Device to Purify DNA from Sputum for Tuberculosis Testing

Background
Tuberculosis (TB) continues to cause significant mortality and morbidity throughout the world, especially in HIV-infected individuals.  Increases in drug resistant TB cases have been occurring in many high endemic countries that have limited resources to identify and diagnose patients. Standard diagnostic techniques for TB include sputum smear microscopy to detect acid fast bacilli and microbiological culture confirmation. As a result, diagnosis of TB is both difficult and time consuming, especially in smear-negative, HIV-infected and pediatric patients.  Molecular technologies are under development for TB case detection and identification of drug resistance mutations in lower levels of the health care system. These technologies will make use of sputum samples, the most important specimen type for TB diagnosis. Processing of sputum prior to PCR amplification will be necessary. A simple, inexpensive device to purify DNA from sputum and re-suspend the DNA in a buffer along with a compatible transfer system to molecular diagnostic assays is being sought. The purified DNA sample from the device should be compatible with many different technologies, thus removing the very difficult step of sample processing from development of the molecular test for Mtb detection and drug resistance testing. This would also allow the sputum processing and molecular testing to be delinked, with processing done immediately at the point-of-care.  
Project goal
The goal of this solicitation is to develop an inexpensive (less than $10), easy to use device for processing sputum samples to obtain purified DNA for TB testing to be used in a rural clinic setting with minimal infrastructure.  Processing of the sputum must be performed in less than 30 minutes, without the need for external electricity (battery power can be proposed).  The resulting material must: 1) be stable over a wide range of ambient temperatures in TB endemic areas (approximately 5 to 50_C), 2) must provide DNA comparable in quality and quantity to DNA prepared using standard laboratory methods , and 3) must provide results comparable to those obtained using DNA from standard laboratory methods in molecular assays for Mtb. 
 
NOTE: The use of TB positive sputum samples may be proposed, but due to the difficulty in obtaining these samples, the use of spiked TB negative sputum samples from donors or artificial sputum samples may also be proposed for initial studies.    
Phase I activities
?       Development of a method for processing sputum to purify DNA for TB testing. 
a.      Sample processing time must be no more than 30 minutes with no more than 2-3 steps performed by the operator.
b.      DNA recovery must be at least 50% compared to a gold standard laboratory method and must allow detection of Mtb in sputum containing at least 5-20 CFU/ml using a standard molecular detection method.
c.      The CV for inter-operator variability must be no more than 20%.
d.      Processed specimens must be stable for at least 7 days at temperatures ranging from 5 to 50_C.
?       Initial evaluation of the product compared to DNA obtained using standard laboratory methods with at least one molecular TB test technology
Phase II activities
?         Production of a small lot to be used for validation testing with at least one molecular Mtb test.  Pre-defined validation targets should be specified.
?         Validation testing with at least one Mtb test, to include precision, accuracy, sensitivity, and specificity with a standard laboratory method of DNA preparation as the comparator
?         Manufacture of the product under good manufacturing practices (GMP) and compliant with the requirements of ISO 13485
 
?         Development of a quality control program to ensure lot-to-lot consistency

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