Methods for detecting cystic fibrosis mutations using mitra tip extraction

Abstract

The present disclosure provides methods for determining whether a patient exhibiting cystic fibrosis symptoms, or a patient at risk for cystic fibrosis, will benefit from treatment with one or more anti-cystic fibrosis therapeutic agents. These methods are based on detecting hereditary cystic fibrosis related mutations in small-volume dried biological fluid samples that are collected using a volumetric absorptive microsampling device. Kits for use in practicing the methods are also provided.

1 . A method for detecting the presence or absence of at least one mutation in a patient sample cystic fibrosis transmembrane regulator (CFTR) nucleic acid comprising
 (a) eluting a dried biological fluid sample from an absorbent tip of a volumetric microsampling device by contacting the absorbent tip of the microsampling device with a lysis buffer and Proteinase K;   (b) extracting the sample CFTR nucleic acid from the dried biological fluid sample eluted from the absorbent tip of a microsampling device;   (c) generating a library comprising amplicons corresponding to a plurality of target segments of the sample CFTR nucleic acid; and   (d) detecting the presence or absence of at least one mutation in at least one of the amplicons in the library using high throughput massive parallel sequencing.   
     
     
         2 . The method of  claim 1 , wherein the dried biological fluid sample is dried plasma, dried serum, or dried whole blood. 
     
     
         3 . The method of  claim 1 , wherein the dried biological fluid sample on the absorbent tip of the microsampling device is collected from a patient via fingerstick. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the lysis buffer comprises guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100. 
     
     
         6 . The method of  claim 1 , wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with the lysis buffer for up to 15 minutes at 90° C. 
     
     
         7 . The method of  claim 1 , wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with Proteinase K for up to 1 hour at 56° C. 
     
     
         8 . The method of  claim 1 , wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with Proteinase K for up to 16-18 hours at 56° C. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the microsampling device is a MITRA® tip. 
     
     
         11 . The method of  claim 1 , wherein the sample volume of the microsampling device is no more than 10-20 μL. 
     
     
         12 . The method of  claim 1 , wherein no more than 400 ng of genomic DNA is eluted from the absorbent tip of the microsampling device. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the at least one mutation is selected from among a base change, a gene deletion and a gene duplication. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the at least one mutation is associated with cystic fibrosis. 
     
     
         17 . The method of any  claim 1 , wherein the dried biological fluid sample is obtained from an individual exhibiting cystic fibrosis symptoms, or having a family history of cystic fibrosis or a CFTR mutation. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the plurality of target segments, together, span all coding and non-coding regions of the CFTR gene. 
     
     
         20 . The method of  claim 19 , wherein the plurality of target segments further span about 1000 nucleotides of a promoter region immediately upstream of the first exon of the CFTR gene. 
     
     
         21 . The method of  claim 20 , wherein the plurality of target segments further span about 200 to 350 nucleotides immediately downstream of the CFTR gene. 
     
     
         22 . The method of  claim 1 , wherein the high throughput massive parallel sequencing is performed using pyrosequencing, reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing, sequencing by synthesis, sequencing by ligation, or SMRT™ sequencing. 
     
     
         23 . The method of  claim 1 , wherein the high throughput massive parallel sequencing involves a read depth approach. 
     
     
         24 - 34 . (canceled) 
     
     
         35 . The method of  claim 1 , further comprising selecting the patient for treatment with an anti-cystic fibrosis therapeutic agent if at least one mutation in at least one of the amplicons in the library is detected. 
     
     
         36 . The method of  claim 35 , wherein the anti-cystic fibrosis therapeutic agent is one or more agents selected from the group consisting of penicillin, amoxicillin, cephalosporins, macrolides, fluoroquinolones, sulfonamides, Tetracyclines, aminoglycosides, colistin, Amcinonide, Betamethosone diproprionate, Clobetasol, Clocortolone, Dexamethasone, Diflorasone, Dutasteride, Flumethasone Pivalate, Flunisolide, Fluocinolone Acetonide, Fluocinonide, Fluorometholone, Fluticasone propionate, Fluticasone propionate, Fluticasone propionate, Flurandrenolide, Hydroflumethiazide, aceclofenac, acemetacin, aspirin, celecoxib, dexibuprofen, dexketoprofen, diclofenac, etodolac, etoricoxib, fenoprofen, flurbiprofen, ibuprofen, indometacin, ketoprofen, mefenamic acid, meloxicam, nabumetone, naproxen, sulindac, tenoxicam, tiaprofenic acid, expectorants, antihistamines, cough suppressants, Dextromethorphan, hypertonic salines, dornase alfa, mucolytics, pancreatic enzymes, vitamin A, vitamin D, vitamin E, vitamin K, and supplements reduce stomach acid.