Point of need testing device and methods of use thereof

Abstract

The present invention relates to the rapid and electricity-free, point-of-care, multiplexed detection and quantification of at least one or more nucleic acid sequences from nucleic acids corresponding to a plurality of pathogens or biomarkers using a micropatterned lateral flow device. Rapid and molecular-level sensitive differential diagnosis of a disease condition may be enabled without the need for a delayed laboratory test so that timely treatment can be administered.

1 . A point of need testing device for detecting the presence of a target nucleic acid sequence in a biological sample from a subject, comprising:
 at least one lateral flow device, wherein said at least one lateral flow device comprises:
 (a) a sample loading area positioned at one end of the lateral flow device, comprising a debris trapping material; 
 (b) an area comprising a detectably labelled probe specific for a target nucleic acid sequence, wherein said detectably labelled probe is not bound to the lateral flow device and is capable of wicking across at least a portion of the lateral flow device; 
 (c) an area comprising a capture probe for the target nucleic acid sequence, wherein said capture probe for the target nucleic acid sequence is immobilized on the lateral flow device; and 
 (d) absorbent material, wherein the absorbent material wicks an aqueous sample across the lateral flow device when the aqueous sample is added to the sample loading area. 
   
     
     
         2 . The point of need testing device of  claim 1 , wherein said debris trapping material comprises glass fiber. 
     
     
         3 . The point of need testing device of  claim 1 , wherein said at least one lateral flow device further comprises an area comprising a second capture probe for a control nucleic acid sequence, wherein said control nucleic acid sequence is complementary to a sequence of the probe specific for the target nucleic acid sequence, and wherein said second capture probe for a control nucleic acid sequence is attached to the lateral flow device. 
     
     
         4 . The point of need testing device of  claim 1 , wherein the detectably labelled probe specific for a target nucleic acid sequence is labeled with a moiety selected from a gold nanoparticle, a protein binding ligand, a hapten, an antigen, a fluorescent compound, a dye, a radioactive isotope and an enzyme. 
     
     
         5 . (canceled) 
     
     
         6 . The point of need testing device of  claim 1 , wherein the capture probe is immobilized on the lateral flow device by covalent coupling. 
     
     
         7 . The point of need testing device of  claim 1 , wherein the capture probe is immobilized on the lateral flow device by affinity binding. 
     
     
         8 . The point of need testing device of  claim 7 , wherein the capture probe is attached to the lateral flow device by biotin: streptavidin affinity binding. 
     
     
         9 . (canceled) 
     
     
         10 . The point of need diagnostic device of  claim 1 , wherein the lateral flow device comprises a solid support. 
     
     
         11 . The point of need diagnostic device of  claim 10 , wherein the solid support is selected from glass, paper, nitrocellulose and thread. 
     
     
         12 . The point of need testing device of  claim 1 , wherein the target nucleic acid sequence is a nucleic acid sequence from a eukaryotic source, a microorganism, a virus, or a microbiome. 
     
     
         13 . The point of need testing device of  claim 12 , wherein the eukaryotic source is selected from algae, protozoa, fungi, slime molds and/or mammalian cells. 
     
     
         14 . (canceled) 
     
     
         15 . The point of need testing device of  claim 12 , wherein the microorganism or virus is selected from  Escherichia, Campylobacter, Clostridium difficile , Enterotoxigenic  E. coli  (ETEC), Enteroaggregative  Escherichia coli  (EAggEC), Shiga-like Toxin producing  E. coli, Salmonella, Shigella, Vibrio cholera, Yersinia enterocolitica , Adenovirus, Norovirus, Rotavirus A,  Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Clostridia , Methicillin-resistant  Staphylococcus aureus  MRSA,  Klebsiella pneumonia , flu, Zika, dengue, chikungunya, West Nile virus, Japanese encephalitis, malaria, HIV, H1N1, and  Clostridium difficile  resistant organisms. 
     
     
         16 . The point of need testing device of  claim 1 , wherein the device comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty, or at least twenty five lateral flow devices. 
     
     
         17 . The point of need testing device of  claim 16 , wherein each lateral flow device comprises probes specific for a different microorganism or virus. 
     
     
         18 . The point of need testing device of  claim 16 , wherein the lateral flow devices are arranged in a radial manner. 
     
     
         19 . The point of need testing device of  claim 18 , wherein the lateral flow devices extend radially from a centrally positioned sample loading area. 
     
     
         20 . The point of need testing device of  claim 16 , wherein the lateral flow devices are arranged in a lateral manner. 
     
     
         21 . A method for detecting the presence of a target nucleic acid sequence a microorganism, or a virus in a biological sample from a subject, comprising:
 (i) lysing cells within the biological sample;   (ii) adding the lysed sample to a sample loading area of a point of need testing device, wherein said point of need testing device comprises: at least one lateral flow device, wherein said at least one lateral flow device comprises:
 (a) a sample loading area positioned at one end of the lateral flow device, comprising a debris trapping material; 
 (b) an area comprising a detectably labelled probe specific for a target nucleic acid sequence, wherein said detectably labelled probe is not bound to the lateral flow device and is capable of wicking across at least a portion of the lateral flow device; 
 (c) an area comprising a capture probe for the target nucleic acid sequence, wherein said capture probe for the target nucleic acid sequence is immobilized on the lateral flow device; and 
 (d) absorbent material, wherein the absorbent material wicks an aqueous sample across the lateral flow device when the aqueous sample is added to the sample loading area; and 
   (iii) detecting a trimolecular hybridization of: (1) the target nucleic acid sequence, (2) the detectably labelled probe specific for the target nucleic acid sequence, and (3) the capture probe for the target nucleic acid sequence.   
     
     
         22 .- 27 . (canceled) 
     
     
         28 . The method of  claim 21 , wherein nucleic acids are not isolated or purified from the lysed sample. 
     
     
         29 . The method of  claim 21 , wherein the biological sample is not subject to any further processing steps prior to or during the steps of the claimed method. 
     
     
         30 . (canceled) 
     
     
         31 . The method according to  claim 21 , wherein the biological sample from the subject is selected from stool, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mammary secretions, mucosal secretion, stool, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. 
     
     
         32 - 50 . (canceled) 
     
     
         51 . The point of need testing device of  claim 1 , wherein the target nucleic acid sequence is an rDNA or rRNA sequence.