Assays for detecting multiple tick-borne pathogens

Abstract

Kits, methods, and material for use in detecting tick-borne microorganism(s) are provided herein. One is able to detect DNA from several different species of microorganisms from samples (bodily fluid, tissue, etc.) from ticks and/or mammals to determine if one or more of the microorganisms has infected the tick and/or mammal. The assay uses PCR techniques with a multiple primers and probes. The microorganisms can be in the Borrelial Layer, the Rickettsiales Layer, and/or the Babesial Layer.

We, the inventors, claim: 
     
         1 . A kit for detecting  Borrelia  spp.,  Rickettsia -like bacteria, or  Babesia  spp. in a sample comprising at least one primer set for detecting said  Borrelia  spp.,  Rickettsia -like bacteria, or  Babesia  spp., an amplification control DNA, instructions for using said at least one primer set and said amplification control plasmid, optionally PCR reagents, and optionally reagents for isolating DNA from a sample. 
     
     
         2 . The kit of  claim 1 , wherein said at least one primer set for said  Borrelia  spp. is selected from the primer sets of:
 (i) SEQ ID NOs: 1 and 2,   (ii) SEQ ID NOs: 5 and 7,   (iii) SEQ ID NOs: 6 and 7,   (iv) SEQ ID NOs: 11 and 13,   (v) SEQ ID NOs: 12 and 13, and   (vi) SEQ ID NOs: 17 and 18.   
     
     
         3 . The kit of  claim 2 , further comprising at least one probe that contains a DNA sequence that present within the amplicon generated by each of said primer sets. 
     
     
         4 . The kit of  claim 3 , wherein said at least one probe comprises SEQ ID NOs: 3, 8, 14, or 19. 
     
     
         5 . The kit of  claim 1 , wherein said at least one primer set for said  Rickettsia -like bacteria is selected from the primer sets of:
 (vii) SEQ ID NOs: 21 and 22,   (viii) SEQ ID NOs: 25 and 26,   (ix) SEQ ID NOs: 29 and 30,   (x) SEQ ID NOs: 33 and 34, and   (xi) SEQ ID NOs: 36 and 34.   
     
     
         6 . The kit of  claim 5 , further comprising at least one probe that contains a DNA sequence that present within the amplicon generated by each of said primer sets. 
     
     
         7 . The kit of  claim 6 , wherein said at least one probe comprises SEQ ID NOs: 23, 27, or 31. 
     
     
         8 . The kit of  claim 1 , wherein said at least one primer set for said  Babesia  spp. is selected from the primer sets of:
 (xii) SEQ ID NOs: 38 and 39, and   (xiii) SEQ ID NOs: 77 and 39.   
     
     
         9 . The kit of  claim 8 , further comprising at least one probe that contains a DNA sequence that present within the amplicon generated by each of said primer sets. 
     
     
         10 . The kit of  claim 9 , wherein said at least one probe comprises SEQ ID NO: 40. 
     
     
         11 . The kit of  claim 1 ; wherein said kit comprises said primer sets:
 (i) SEQ ID NOs: 1 and 2,   (ii) SEQ ID NOs: 5 and 7,   (iii) SEQ ID NOs: 6 and 7,   (iv) SEQ ID NOs: 11 and 13,   (v) SEQ ID NOs: 12 and 13,   (vi) SEQ ID NOs: 17 and 18,   (vii) SEQ ID NOs: 21 and 22,   (viii) SEQ ID NOs: 25 and 26,   (ix) SEQ ID NOs: 29 and 30,   (x) SEQ ID NOs: 33 and 34,   (xi) SEQ ID NOs: 36 and 34,   (xii) SEQ ID NOs: 38 and 39, and   (xiii) SEQ ID NOs: 77 and 39.   
     
     
         12 . The kit of  claim 11 , wherein said  Borrelia  spp. is selected from the group consisting of  Borrelia burgdorferi, B. hermsii, B. turicatae , and  B. parkeri ; wherein said  Rickettsia -like bacteria is selected from the group consisting of  Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum , and  Rickettsia rickettsii ; and wherein said  Babesia  spp. is selected from the group consisting of  Babesia canis vogeli, B. gibsoni, B. canis canis, B. canis rossi, B. caballi, B. odocoilei, B. divergens , and  B. bigemina.    
     
     
         13 . The kit of  claim 11 , wherein said kit further comprises at least one  Borrelia  spp. probe, at least one  Rickettsia -like bacteria probe, and at least one  Babesia  spp. probe; wherein said at least one  Borrelia  spp. probe comprises at least one of SEQ ID NOs: 3, 8, 14, and 19; wherein said at least one  Rickettsia -like bacteria probe comprises at least one of SEQ ID NOs: 23, 27, and 31; and wherein said at least  Babesia  spp. probe comprises SEQ ID NO: 40. 
     
     
         14 . The kit of  claim 11 , wherein said primer sets further comprise
 (xiv) SEQ ID NOs: 46 and 47,   (xv) SEQ ID NOs: 49 and 50,   (xvi) SEQ ID NOs: 52 and 53,   (xvii) SEQ ID NOs: 56 and 57,   (xviii) SEQ ID NOs: 60 and 61,   (xix) SEQ ID NOs: 64 and 65; and wherein said  Borrelia  spp. is selected from the group consisting of  Borrelia burgdorferi, B. hermsii, B. turicatae , and  B. parkeri ; wherein said  Rickettsia -like bacteria is selected from the group consisting of  Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum , and  Rickettsia rickettsii ; and wherein said  Babesia  spp. is selected from the group consisting of  Babesia canis vogeli, B. gibsoni, B. canis canis, B. canis rossi, B. caballi, B. odocoilei, B. divergens, B. bigemina , and  B. conradae.      
     
     
         15 . The kit of  claim 14 , wherein said kit further comprises at least one  Borrelia  spp. probe, at least one  Rickettsia -like bacteria probe, and at least one  Babesia  spp. probe; wherein said at least one  Borrelia  spp. probe comprises at least one of SEQ ID NOs: 3, 8, 14, and 19; wherein said at least one  Rickettsia -like bacteria probe comprises at least one of SEQ ID NOs: 23, 27, and 31; and wherein said at least  Babesia  spp. probe comprises at least one of SEQ ID NOs: 40, 54, 58, 62, and 66. 
     
     
         16 . The kit of  claim 1 , wherein said amplification control plasmid comprises SEQ ID NO: 68. 
     
     
         17 . The kit of  claim 1 , wherein when said sample is obtained from a canine, said amplification control plasmid comprises SEQ ID NO: 45, and said kit further comprises canine specific primer set of SEQ ID NOs: 42 and 43 and a canine specific probe comprising SEQ ID NO: 44. 
     
     
         18 . The kit of  claim 1 , wherein said  Borrelia  spp. is  B. burgdorferi ; wherein said  Rickettsia -like bacteria is  E. canis, E. chaffeensis , and  A. phagocytophilum;    wherein said primer set for detecting said  B. burgdorferi  comprises at least one of
 (a)(i) SEQ ID NOs: 69 and 70 and (a)(ii) SEQ ID NOs: 1 and 2; 
   wherein said primer set for detecting  E. canis  comprises at least one of
 (b)(i) SEQ ID NOs: 71 and 72 and (b)(ii) SEQ ID NOs: 29 and 30; 
   wherein said primer set for detecting  E. chaffeensis  comprises at least one of
 (c)(i) SEQ ID NOs: 73 and 74 and (c)(ii) SEQ ID NOs: 33 and 34; and 
   wherein said primer set for detecting  A. phagocytophilum  comprises at least one of
 (d)(i) SEQ ID NOs: 75 and 76 and (d)(ii) SEQ ID NOs: 25 and 26. 
   
     
     
         19 . The kit of  claim 18 , wherein when said sample is obtained from a canine, said amplification control plasmid comprises SEQ ID NO: 45, and said kit further comprises canine specific primer set of SEQ ID NOs: 42 and 43. 
     
     
         20 . A method for detecting at least one tick-borne pathogen in a sample from an animal comprising
 a) optionally, isolating DNA from said sample to provided isolated DNA;   b) contacting said isolated DNA with a plurality of primer sets and DNA polymerase to provide a first layer PCR mix, wherein said plurality of primer sets detect at least one tick-borne pathogen;   c) performing PCR on said first layer PCR mix to generate at least one amplicon; and   d) determining the identify of said generated at least one amplicon, wherein said at least one amplicon is specific for detecting said at least one tick-borne pathogen in said sample.   
     
     
         21 . The method of  claim 20 , wherein said animal from which said sample is obtained is a tick or a mammal. 
     
     
         22 . The method of  claim 20 , wherein said at least one tick-borne pathogen is a  Borrelia  spp.,  Rickettsia -like bacteria, or a  Babesia  spp.; wherein said primer sets for detecting said  Borrelia  spp. is at least one of said primer sets of:
 (i) SEQ ID NOs: 1 and 2,   (ii) SEQ ID NOs: 5 and 7,   (iii) SEQ ID NOs: 6 and 7,   (iv) SEQ ID NOs: 11 and 13,   (v) SEQ ID NOs: 12 and 13, and   (vi) SEQ ID NOs: 17 and 18;   
       wherein said primer sets for detecting said  Rickettsia -like bacteria is at least one of said primer sets of:
 (vii) SEQ ID NOs: 21 and 22, 
 (viii) SEQ ID NOs: 25 and 26, 
 (ix) SEQ ID NOs: 29 and 30, 
 (x) SEQ ID NOs: 33 and 34, and 
 (xi) SEQ ID NOs: 36 and 34; 
 
       and wherein said primer set for detecting said  Borrelia  spp. is at least one of the primer sets of:
 (xii) SEQ ID NOs: 38 and 39, and 
 (xiii) SEQ ID NOs: 77 and 39. 
 
     
     
         23 . The method of  claim 22 , wherein said  Borrelia  spp. is selected from the group consisting of  Borrelia burgdorferi, B. hermsii, B. turicatae , and  B. parkeri ; wherein said  Rickettsia -like bacteria is selected from the group consisting of  Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum , and  Rickettsia rickettsii ; and wherein said  Babesia  spp. is selected from the group consisting of  Babesia canis vogeli, B. gibsoni, B. canis canis, B. canis rossi, B. caballi, B. odocoilei, B. divergens , and  B. bigemina.    
     
     
         24 . The method of  claim 22 , wherein said first layer PCR mix further comprises at least one probe to detect  Borrelia  spp., at least one probe to detect  Rickettsia -like bacteria probe, and at least one probe to detect  Babesia  spp.; wherein said at least one  Borrelia  spp. probe comprises at least one of SEQ ID NOs: 3, 8, 14, and 19; wherein said at least one  Rickettsia -like bacteria probe comprises at least one of SEQ ID NOs: 23, 27, and 31; and wherein said at least  Babesia  spp. probe comprises SEQ ID NO: 40. 
     
     
         25 . The method of  claim 22 , wherein said tick-borne pathogen is  Borrelia  spp., said method further comprises
 e) contacting said isolated DNA with at least one  Borrelia  spp. primer sets and DNA polymerase to provide a second layer PCR mix;   f) performing PCR on said second layer PCR mix to generate at least one amplicon; and   g) determining the identify of said generated at least one amplicon, wherein said at least one  Borrelia  spp. primer sets detect  B. burgdorferi, B. hermsii, B. turicatae , or  B. parkeri      wherein said  B. burgdorferi  primer set is (i) SEQ ID NOs: 1 and 2;   wherein said  B. hermsii  set is at least one of (ii) SEQ ID NOs: 5 and 7, (iii) SEQ ID NOs: 6 and 7, (xiv) SEQ ID NOs: 46 and 47, and (xv) SEQ ID NOs: 49 and 50;   wherein said  B. turicatae  is at least one of (iv) SEQ ID NOs: 11 and 13 and (v) SEQ ID NOs: 12 and 13; and   wherein said  B. parkeri  primer set is (vi) SEQ ID NOs: 17 and 18.   
     
     
         26 . The method of  claim 25 , wherein said second layer PCR mix further comprises at least one probe to detect  B. burgdorferi, B. hermsii, B. turicatae , or  B. parkeri ; wherein said  B. burgdorferi  probe comprises SEQ ID NO: 3; wherein said  B. hermsii  probe comprises SEQ ID NO: 8; wherein said  B. turicatae  probe comprises SEQ ID NO: 14; and wherein said  B. parkeri  probe comprises SEQ ID NO: 19. 
     
     
         27 . The method of  claim 22 , wherein said tick-borne pathogen is  Rickettsia -like bacteria, said method further comprises
 e) contacting said isolated DNA with at least one  Rickettsia -like bacteria primer sets and DNA polymerase to provide a second layer PCR mix;   f) performing PCR on said second layer PCR mix to generate at least one amplicon; and   g) determining the identify of said generated at least one amplicon; wherein said at least one  Rickettsia -like bacteria primer sets detect  Rickettsia rickettsii, Anaplasma phagocytophilum, Ehrlichia canis, E. chaffeensis , or  E. ewingii;      wherein said  R. rickettsii  primer set is (vii) SEQ ID NOs: 21 and 22;   wherein said  A. phagocytophilum  primer set is (viii) SEQ ID NOs: 25 and 26;   wherein said  E. canis  primer set is (ix) SEQ ID NOs: 29 and 30;   wherein said  E. chaffeensis  primer set is (x) SEQ ID NOs: 33 and 34; and   wherein said  E. ewingii  primer set is (xi) SEQ ID NOs: 36 and 34.   
     
     
         28 . The method of  claim 27 , wherein said second layer PCR mix further comprises at least one probe to detect  R. rickettsii, A. phagocytophilum, E. canis, E. chaffeensis , or  E. ewingii ; wherein said  R. rickettsii  probe comprises SEQ ID NO: 23; wherein said  A. phagocytophilum  probe comprises SEQ ID NO: 27; and wherein said  E. canis  probe,  E. chaffeensis , and  E. ewingii  comprise SEQ ID NO: 31. 
     
     
         29 . The method of  claim 22 , wherein said tick-borne pathogen is  Babesia  spp., said method further comprises
 e) contacting said isolated DNA with at least one  Babesia  primer sets and DNA polymerase to provide a second layer PCR mix;   f) performing PCR on said second layer PCR mix to generate at least one amplicon; and   g) determining the identify of said generated at least one amplicon, wherein said at least one  Babesia  primer sets detect  Babesia canis vogeli, B. gibsoni , or  B. conradae;      wherein said  B. canis vogeli  primer set is at least one of (xvi) SEQ ID NOs: 52 and 53 and (xvii) SEQ ID NOs: 56 and 57;   wherein said  B. gibsoni  primer set is (xviii) SEQ ID NOs: 60 and 61; and   wherein said  B. conradae  primer set is (xix) SEQ ID NOs: 64 and 65.   
     
     
         30 . The method of  claim 29 , wherein said second layer PCR mix further comprises at least one probe to detect  B. canis vogeli, B. gibsoni , or  B. conradae ; wherein said  B. canis vogeli  probe comprises at least one of SEQ ID NO: 54 and SEQ ID NO: 58; wherein said  B. gibsoni  probe comprises SEQ ID NO: 62; and wherein said  B. conradae  probe comprises SEQ ID NO: 66. 
     
     
         31 . The method of  claim 20 , wherein said at least one tick-borne pathogen is selected from the group consisting of  B. burgdorferi, E. canis, E. chaffeensis , and  A. phagocytophilum ; wherein said plurality of primer sets comprise a  B. burgdorferi  primer set, an  E. canis  primer set, an  E. chaffeensis  primer set, and an  A. phagocytophilum  primer set;
 wherein said  B. burgdorferi  primer set comprises SEQ ID NOs: 69 and 70 or SEQ ID NOs: 1 and 2;   wherein said  E. canis  primer set comprises SEQ ID NOs: 71 and 72 or SEQ ID NOs: 29 and 30;   wherein  E. chaffeensis  primer set comprises SEQ ID NOs: 73 and 74 or SEQ ID NOs: 33 and 34; and   wherein said  A. phagocytophilum  primer set comprises SEQ ID NOs: 75 and 76 or SEQ ID NOs: 25 and 26.   
     
     
         32 . The method of  claim 31 , wherein said step (d) determining the identify of said generated at least one amplicon comprising running said amplicon on a gel and determining the size of said amplicon; wherein when said amplicon is 235 bp or 75 bp,  B. burgdorferi  is present in said sample; wherein when said amplicon is 389 bp or 113 bp,  E. canis  is present in said sample; wherein when said amplicon is 390 bp or 111 bp,  E. chaffeensis  is present in said sample; and wherein when said amplicon is 644 bp or 95 bp,  A. phagocytophilum  is present in said sample. 
     
     
         33 . A plasmid comprising SEQ ID NO: 68.