US2005170341A1PendingUtilityA1
Methods for detecting Ehrlichia canis and Ehrlichia chaffensis in vertebrate and invertebrate hosts
Priority: Aug 25, 2000Filed: May 2, 2002Published: Aug 4, 2005
Est. expiryAug 25, 2020(expired)· nominal 20-yr term from priority
C12Q 1/689
48
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Claims
Abstract
Tools and methods for detecting the presence of E. canis and E. chaffeensis in a sample obtained from an animal are provided. The methods employ a polymerase chain reaction and primer sets that are based on the p30 gene of E. canis and the p28 gene of E. chaffeensis. The present invention also relates to the p30 and the p28 primer sets. Each p30 primer set comprises a first primer and the second primer, both of which are from 15 to 35 nucleotides in length. The first p30 primer comprises a sequence which is complementary to a consecutive sequence, within the following sequence: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30 primer comprises a sequence which is complementary to the inverse complement of a consecutive sequence contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2. The first p28 comprises a sequence which is complementary to a consecutive sequenc, within the following sequence: A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO. 3. The second p28 primercomprises a sequence which is complementary to the inverse complement of a consecutive sequencewithin one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO. 4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO. 5.
Claims
exact text as granted — not AI-modified1 . A method for detecting E. canis in a sample obtained from an animal, comprising
(a) providing a primer set comprising:
(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within the following sequence:
SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT,.;
(b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the length or sequence of the PCR products of step (b), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. canis p30 gene which is located between the regions to which the forward primer and the reverse primer bind is indicative of the presence of E. canis in the sample.
2 . The method of claim 1 wherein the consecutive sequence is at least 14 nucleotides in length.
3 . The method of claim 1 wherein the forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:
PAIR 1:
ATAAACACGCTGACTTTACTGTTCC, S,
SEQ ID NO. 6
GTGATGAGATAGAGCGCAGTACC,.;
SEQ ID NO. 7
PAIR 2
AACACGCTGACTTTACTGTTCC,,
SEQ ID NO. 8
ATGGCTGCCGATGTGTGATG,,
SEQ ID NO. 9
PAIR 3:
ACGCTGACTTTACTGTTCCAAAC,,
SEQ ID NO. 10
ATGGCTGCCGATGTGTGATG,,
SEQ ID NO. 11
PAIR 4:
AACATGATTGGGATGGAAGTC,,
SEQ ID NO. 12
GCCGATGTGTGATGAGATAG,;
SEQ ID NO. 13
PAIR 5:
AAACATGATTGGGATGGAAGTC,,
SEQ. ID NO. 12
GCCGATGTGTGATGAGATAGAG,;
SEQ ID NO. 14
PAIR 6:
GATTGGGATGGAAGTCCAATAC,,
SEQ. ID. NO. 13
GCCGATGTGTGATGAGATAG,;
SEQ ID NO. 11
PAIR 7:
GATTGGGATGGAAGTCCAATAC,,
SEQ ID NO. 13
GCCGATGTGTGATGAGATAGAG,;
SEQ ID NO. 14
PAIR 8:
ACACGCTGACTTTACTGTTCCAAAC,;
SEQ ID NO. 15
ATGGCTGCCGATGTGTGATGAG,;
SEQ ID NO. 16
PAIR 9:
GTGTCTCACATTTTGGTAGCTTCTC,,
SEQ ID NO. 17
CTTGGGCCACCCATTGAGTAAC,;
SEQ ID NO. 18
PAIR 10
GATTGGGATGGAAGTCCAATAC,,
SEQ. ID. NO. 13
CGATGTGTGATGAGATAGAGC,;
SEQ ID NO. 19
PAIR 11:
TGATTGGGATGGAAGTCCAATAC,,
SEQ ID NO. 20
CGATGTGTGATGAGATAGAGCG,,
SEQ ID NO. 21
PAIR 12:
CATGATTGGGATGGAAGTCCAATAC,,
SEQ ID NO. 21
ATGGCTGCCGATGTGTGATG,;
SEQ ID NO. 11
PAIR 13:
CCAAGTGTCTCACATTTTGGTAGC,,
SEQ ID NO. 22
TGGGCCACCCATTGAGTAAC,.
SEQ ID NO. 23
4 . The method of claim 3 wherein the primer set is pair 12.
5 . The method of claim 3 wherein the primer set is pair 1.
6 . A method for detecting E. canis in a sample obtained from an animal, comprising
(a) providing a first primer set comprising:
(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within the following sequence:
SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT,.;
(b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction to provide a pool of PCR products, (c) amplifying the products of step (b) using a polymerase chain reaction and a second primer set comprising:
(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within the following sequence:
SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT,.;
wherein one or both of the primers of the second primer set are internal to the primers of the first primer set; and
(d) determining the length or sequence of the PCR products of step (c), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. canis p30 gene which is located between the regions to which the forward primer of the second primer set and the reverse primer of the second primer set bind is indicative of the presence of E. canis in the sample.
7 . A method for detecting E. chaffeensis in a sample obtained from an animal, comprising
(a) providing a primer set comprising:
(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO.3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within one of the following two sequences:
SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,
(b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the length or sequence of the PCR products of step (b), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. chaffeensis p28 gene which is located between the regions to which the forward primer and the reverse primer bind is indicative of the presence of E. chaffeensis in the sample.
8 . The method of claim 7 wherein the consecutive sequence is at least 14 nucleotides in length.
9 . The method of claim 7 wherein the forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:
10 . The method of claim 9 wherein the primer set is pair 1.
11 . The method of claim 9 wherein the primer set is pair 3.
12 . A method for detecting E. chaffeensis in a sample obtained from an animal, comprising
(a) providing a first primer set comprising:
providing a primer set comprising:
(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO. 3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within one of the following two sequences:
SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,
(b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction to provide a pool of PCR products, (c) amplifying the products of step (b) using a polymerase chain reaction and a second primer set comprising:
(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO. 3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within one of the following two sequences:
SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,
wherein one or both of the primers of the second primer set are internal to the primers of the first primer set; and
(d) determining the length or sequence of the PCR products of step (c), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. chaffeensis p28gene which is located between the regions to which the forward primer of the second primer set and the reverse primer of the second primer set bind is indicative of the presence of E. chaffeensis in the sample.
13 . A primer set for detecting E. canis in a sample, said primer set comprising:
(a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence: SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and (ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within the following sequence: SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT,.;
14 . The primer set of claim 13 wherein forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:
PAIR 1:
ATAAACACGCTGACTTTACTGTTCC, S,
SEQ ID NO. 6
GTGATGAGATAGAGCGCAGTACC,.;
SEQ ID NO. 7
PAIR 2
AACACGCTGACTTTACTGTTCC,,
SEQ ID NO. 8
ATGGCTGCCGATGTGTGATG,,
SEQ ID NO. 9
PAIR 3:
ACGCTGACTTTACTGTTCCAAAC,,
SEQ ID NO. 10
ATGGCTGCCGATGTGTGATG,,
SEQ ID NO. 11
PAIR 4:
AACATGATTGGGATGGAAGTC,,
SEQ ID NO. 12
GCCGATGTGTGATGAGATAG,,
SEQ ID NO. 13
PAIR 5:
AAACATGATTGGGATGGAAGTC,,
SEQ. ID NO. 12
GCCGATGTGTGATGAGATAGAG,;
SEQ ID NO. 14
PAIR 6:
GATTGGGATGGAAGTCCAATAC,,
SEQ. ID. NO. 13
GCCGATGTGTGATGAGATAG,;
SEQ ID NO. 11
PAIR 7:
GATTGGGATGGAAGTCCAATAC,,
SEQ ID NO. 13
GCCGATGTGTGATGAGATAGAG,;
SEQ ID NO. 14
PAIR 8:
ACACGCTGACTTTACTGTTCCAAAC,;
SEQ ID NO. 15
ATGGCTGCCGATGTGTGATGAG,;
SEQ ID NO. 16
PAIR 9:
GTGTCTCACATTTTGGTAGCTTCTC,,
SEQ ID NO. 17
CTTGGGCCACCCATTGAGTAAC,;
SEQ ID NO. 18
PAIR 10
GATTGGGATGGAAGTCCAATAC,,
SEQ. ID. NO. 13
CGATGTGTGATGAGATAGAGC,;
SEQ ID NO. 1;
PAIR 11:
TGATTGGGATGGAAGTCCAATAC,,
SEQ ID NO. 20
CGATGTGTGATGAGATAGAGCG,,
SEQ ID NO. 21
PAIR 12:
CATGATTGGGATGGAAGTCCAATAC,,
SEQ ID NO. 21
ATGGCTGCCGATGTGTGATG,;
SEQ ID NO. 11
PAIR 13:
CCAAGTGTCTCACATTTTGGTAGC,,
SEQ ID NO. 22
TGGGCCACCCATTGAGTAAC,.
SEQ ID NO. 23
15 . The primer set of claim 14 wherein the primer set comprises pair 1.
16 . The primer set of claim 14 wherein the primer set comprises pair 12.
17 . A primer set for detecting E. chaffeensis in a sample, said primer set comprising a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:
SEQ ID NO. 3
(a)
A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC
TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT
TAACGG,;
and
+P1 (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within one of the following two sequences:
SEQ ID NO. 4
CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG
TCCAAGAATA GAGCTTGAAG TATCTTATGA,,
or
SEQ ID NO. 5
C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,.
18 . The primer set of claim 17 wherein the forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:
PAIR 1
AGGTAGTGGTATTAACGG,
SEQ ID NO 24
AGATACTTCAAGCTCTATTC,;
SEQ ID NO. 25
PAIR 2:
AGGTAGTGGTATTAACGG,,
SEQ ID NO
TCATAAGATACTTCAAGCTC,;
SEQ ID NO
PAIR 3
CTTCTCTACCTGGAGTATC,,
SEQ ID NO
GCTTATAGAGTAGCTTAAACC,;
SEQ ID NO
PAIR 4:
CAGGTAGTGGTATTAACG,,
SEQ ID NO
CATAAGATACTTCAAGCTC,;
SEQ ID NO
PAIR 5
CAGGTAGTGGTATTAACG,,
SEQ ID NO
GATACTTCAAGCTCTATTC,;
SEQ ID NO
PAIR 6
CTTCTCTACCTGGAGTATC,,
SEQ ID NO
GCTTATAGAGTAGCTTAAAC,;
SEQ ID NO
PAIR 7
CTACCTGGAGTATCATTTTC,,
SEQ ID NO
GGCTTATAGAGTAGCTTAAAC,;
SEQ ID NO
PAIR 8
AATATCTTCTCTACCTGG,,
SEQ ID NO
GATACTTCAAGCTCTATTC,;
SEQ ID NO
PAIR 9
AGTTTTCATAACAAGTGC,,
SEQ ID NO.
CATAAGATACTTCAAGCTC,;
SEQ ID NO
PAIR 10:
AGTTTTCATAACAAGTGC,,
SEQ ID NO
GATACTTCAAGCTCTATTC,;
SEQ ID NO
PAIR 11
CTTCTCTACCTGGAGTATCATTTTC,
SEQ ID NO
GAGTAGCTTAAACCTAACTTTCCTTG,;
SEQ ID NO
PAIR 12:
CTTCTCTACCTGGAGTATC,,
SEQ ID NO
GAGTAGCTTAAACCTAACTTTC,;
SEQ ID NO
PAIR 13
AATATCTTCTCTACCTGG,
SEQ ID NO
TCATAAGATACTTCAAGC,;
SEQ ID NO
PAIR 14:
AATATCTTCTCTACCTGG,
SEQ ID NO
64 CATAAGATACTTCAAGCTC,;
SEQ ID NO
PAIR 15
AATATCTTCTCTACCTGG,
SEQ ID NO
CATAAGATACTTCAAGCTC,;
SEQ ID NO
PAIR 16
CTCTACCTGGAGTATCATTTTC,
SEQ ID NO
GGCTTATAGAGTAGCTTAAACC;
SEQ ID NO
PAIR 17:
CTCTACCTGGAGTATCATTTTC,
SEQ ID NO
GAGTAGCTTAAACCTAACTTTC;
SEQ ID NO
PAIR 18:
ACCTGGAGTATCATTTTC,
SEQ ID NO
TCTGGGCTTATAGAGTAG,;
SEQ ID NO
PAIR 19
CTTCTCTACCTGGAGTATC,
SEQ ID NO
CTGGGCTTATAGAGTAGC,;
SEQ ID NO
PAIR 20
CTTCTCTACCTGGAGTATC,
SEQ ID NO
CTGGGCTTATAGAGTAGC,
SEQ ID NO
19 . The primer set of claim 18 wherein the primer set is pair 1.
20 . The primer set of claim 18 wherein the primer set is pair 3.Join the waitlist — get patent alerts
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