Targeted whole genome amplification method for identification of pathogens

Abstract

The methods disclosed herein relate to methods and compositions for amplifying nucleic acid sequences, more specifically, from nucleic acid sequences of pathogens by targeted whole genome amplification.

What is claimed is: 
     
         1 . A method comprising:
 amplifying at least one pathogen genome from a sample suspected of comprising at least one pathogen genome and at least one background genome using a plurality of targeted whole genome amplification primers, thereby elevating the quantity of nucleic acid representing said at least one pathogen genome relative to the quantity of nucleic acid representing said at least one background genome, wherein said plurality of targeted whole genome amplification primers is selected by:
 i. identifying at least one pathogen genome; 
 ii. identifying at least one background genome; 
 iii. identifying a plurality of genome sequence segments having unique sequences within said pathogen genome sequence; 
 iv. determining frequency of occurrence of members of said plurality of genome sequence segments within said pathogen genome sequence and determining frequency of occurrence of said plurality of genome sequence segments within said background genome sequences; 
 v. calculating a selectivity ratio for said members by dividing said frequency of occurrence within said pathogen genome sequence by said frequency of occurrence of said plurality of genome sequence segments within said background genome sequences; 
 vi. selecting a selectivity ratio threshold value, thereby defining a first sub-set of said plurality of genome sequence segments having selectivity ratios equal to or greater than said selectivity ratio threshold value; 
 vii. determining the lengths of pathogen genome sequence occurring between genome sequence segments of said first sub-set; 
 viii. selecting a second sub-set of genome sequence segments from said first sub-set wherein members of said second sub-set have a mean separation distance of less than a selected length of nucleobases; and 
 ix. selecting targeted whole genome amplification primers that hybridize to members of said second sub-set of genome sequence segments such that, under whole genome amplification conditions, said at least one pathogen genome is amplified selectively over said at least one background genomes. 
   
     
     
         2 . The method of  claim 1  further comprising the step of producing one or more amplification products representing bioagent identifying amplicons from said amplified pathogen genome using one or more primer pairs. 
     
     
         3 . The method of  claim 2  further comprising the step of measuring molecular masses of said amplification products by mass spectrometry. 
     
     
         4 . The method of  claim 3  wherein said mass spectrometry is electrospray time-of-flight mass spectrometry. 
     
     
         5 . The method of  claim 3  further comprising the step of comparing said molecular masses with a database comprising molecular masses of bioagent identifying amplicons of pathogens produced with said primer pairs, thereby identifying said pathogen in said sample. 
     
     
         6 . The method of  claim 3  further comprising the step of calculating base compositions of said amplification products from said molecular masses. 
     
     
         7 . The method of  claim 6  further comprising the step of comparing said base compositions with a database comprising base compositions of bioagent identifying amplicons of pathogens produced with said primer pairs, thereby identifying said pathogen in said sample. 
     
     
         8 . The method of  claim 2  wherein said amplification products are generated using a plurality of primer pairs that define bioagent identifying amplicons. 
     
     
         9 . The method of  claim 8  wherein said plurality of primer pairs are used in a multiplex reaction to generate a plurality of amplification products. 
     
     
         10 . The method of  claim 8  wherein said plurality of primer pairs comprises at least two primer pairs from the group consisting of primer pair numbers: 346 (SEQ ID NOs: 594:602), 348 (SEQ ID NOs: 595:603), 349 (SEQ ID NOs: 596:604), 354 (SEQ ID NOs: 597:605), 358 (SEQ ID NOs: 598:606), 359 (SEQ ID NOs: 599:607), 3346 (SEQ ID NOs: 616:631), 449 (SEQ ID NOs: 600:608), 3350 (SEQ ID NOs: 614:629), 2249 (SEQ ID NOs: 601:609), 3361 (SEQ ID NOs: 620:635), and 3360 (SEQ ID NOs: 612:627). 
     
     
         11 . The method of  claim 8  wherein said plurality of primer pairs comprises primer pair numbers: 346 (SEQ ID NOs: 594:602), 348 (SEQ ID NOs: 595:603), 349 (SEQ ID NOs: 596:604), 3346 (SEQ ID NOs: 616:631). 
     
     
         12 . The method of  claim 8  wherein said plurality of primer pairs comprises primer pair numbers: 346 (SEQ ID NOs: 594:602), 348 (SEQ ID NOs: 595:603), 349 (SEQ ID NOs: 596:604), and 3361 (SEQ ID NOs: 620:635). 
     
     
         13 . The method of  claim 8  wherein said plurality of primer pairs comprises primer pair numbers 346 (SEQ ID NOs: 594:602), 348 (SEQ ID NOs: 595:603), 349 (SEQ ID NOs: 596:604) and at least one of the primer pairs selected from the group consisting of 354 358 (SEQ ID NOs: 598:606), 359 (SEQ ID NOs: 599:607), 3346 (SEQ ID NOs: 616:631), 449 (SEQ ID NOs: 600:608), 3350 (SEQ ID NOs: 614:629), 3361 (SEQ ID NOs: 620:635), and 3360 (SEQ ID NOs: 612:627). 
     
     
         14 . The method of  claim 1  wherein a high processivity polymerase enzyme is used at said amplification step. 
     
     
         15 . The method of  claim 14  wherein said high processivity polymerase enzyme is a recombinant polymerase enzyme. 
     
     
         16 . The method of  claim 14  wherein said high processivity polymerase enzyme is a genetically engineered polymerase enzyme. 
     
     
         17 . The method of  claim 14  wherein said high processivity polymerase enzyme is phi29. 
     
     
         18 . The method of  claim 1 , wherein said sample comprises human whole blood. 
     
     
         19 . The method of  claim 18  further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step. 
     
     
         20 . The method of  claim 1  wherein said sample comprises human buffy coat.