US2014162260A1PendingUtilityA1
Primers, snp markers and method for genotyping mycobacterium tuberculosis
Assignee: NAT HEALTH RESEARCH INSTITUTESPriority: Nov 26, 2012Filed: Nov 26, 2013Published: Jun 12, 2014
Est. expiryNov 26, 2032(~6.3 yrs left)· nominal 20-yr term from priority
C12Q 2600/156C12Q 2600/16C12Q 1/689C12Q 2600/172
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Abstract
The present application provides a primer set for genotyping M. tuberculosis selected from the group consisting of primer sets 1-25 (SEQ ID Nos. 1-50), and also provides an extension primer for genotyping M. tuberculosis selected from one of the group consisting of SEQ ID Nos. 51-75. The present application provides a combination of single-nucleotide polymorphism markers of M. tuberculosis . Further, the present application provides a method and a kit for genotyping M. tuberculosis.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A primer set for genotyping Mycobacterium tuberculosis selected from one of the group consisting of:
Primer set 1:
(SEQ ID No. 1)
ACGTTGGATGTTCTGGACGACCTGTCCTAC
and
(SEQ ID No. 2)
ACGTTGGATGAGCTGCGCCAAGGTTCGTG,
Primer set 2:
(SEQ ID No. 3)
ACGTTGGATGTTGTAGCTGCCCAAATTGCC
and
(SEQ ID No. 4)
ACGTTGGATGGGCTTCAATCTCGGCTTGG,
Primer set 3:
(SEQ ID No. 5)
ACGTTGGATGTATTCAACACCGGCATCGGG
and
(SEQ ID No. 6)
ACGTTGGATGTCGCCTGGTCGTGGAAGAAC,
Primer set 4:
(SEQ ID No. 7)
ACGTTGGATGATCGGACAGCAGAAGGCAC
and
(SEQ ID No. 8)
ACGTTGGATGACTCCCGCGGAACGTGGTG,
Primer set 5:
(SEQ ID No. 9)
ACGTTGGATGCAACACCGGCAACTTCAAC
and
(SEQ ID No. 10)
ACGTTGGATGAATTAGCGTCTCCTCCGTTG,
Primer set 6:
(SEQ ID No. 11)
ACGTTGGATGTCGAACCCGCCGACAAATG
and
(SEQ ID No. 12)
ACGTTGGATGTCGATTGGTCGCATGCACTG,
Primer set 7:
(SEQ ID No. 13)
ACGTTGGATGAAACCTCGGCATAGGGATCG
(SEQ ID No. 14)
ACGTTGGATGTCGACAGGACTATTGGTAGC,
and
Primer set 8:
(SEQ ID No. 15)
ACGTTGGATGAAGACGACGGGCCGGATATG
and
(SEQ ID No. 16)
ACGTTGGATGCGTCAAGAGCTTCCCAAATC,
Primer set 9:
(SEQ ID No. 17)
ACGTTGGATGCATCCGGGAACACCGTAAAC
and
(SEQ ID No. 18)
ACGTTGGATGATCACCTTCTTATCGGGTGG,
Primer set 10:
(SEQ ID No. 19)
ACGTTGGATGCCTGGATTTCAGATATTGCC
and
(SEQ ID No. 20)
ACGTTGGATGTGGCCAGCCCTAGCAAGTC,
Primer set 11:
(SEQ ID No. 21)
ACGTTGGATGAGAACAAACGCGGGATTCAC
and
(SEQ ID No. 22)
ACGTTGGATGTCTCCCGGAGATCACCATTC,
Primer set 12:
(SEQ ID No. 23)
ACGTTGGATGGTTGTTTTTGGCCGGGCAG
and
(SEQ ID No. 24)
ACGTTGGATGATCGAGCAGACTCAGCGCTT,
Primer set 13:
(SEQ ID No. 25)
ACGTTGGATGTGCTACCGCCAATGTTCAAC
and
(SEQ ID No. 26)
ACGTTGGATGATGGCGTTGACATAACTCGG,
Primer set 14:
(SEQ ID No. 27)
ACGTTGGATGATAGCAAGCACGATTGCGAC
and
(SEQ ID No. 28)
ACGTTGGATGACCCCCCGCTGAGGGCGTA,
Primer set 15:
(SEQ ID No. 29)
ACGTTGGATGGATTCGATTGGGGAAACGGC
and
(SEQ ID No. 30)
ACGTTGGATGTTCCACATTGGTGATCAGCG,
Primer set 16:
(SEQ ID No. 31)
ACGTTGGATGCAAACGGCGTCACTTTGGTC
and
(SEQ ID No. 32)
ACGTTGGATGTGAAATGTGGGCCCAAGACG,
Primer set 17:
(SEQ ID No. 33)
ACGTTGGATGCGATTTCGATCGGGATGTTG
and
(SEQ ID No. 34)
ACGTTGGATGCAATCACGATCCCCTCAATC,
Primer set 18:
(SEQ ID No. 35)
ACGTTGGATGAGGCAAAGGAAAATCGACCG
and
(SEQ ID No. 36)
ACGTTGGATGTTGACAAACTGAAACACCGC,
Primer set 19:
(SEQ ID No. 37)
ACGTTGGATGACAACCGGCCGCAGCGTTT
and
(SEQ ID No. 38)
ACGTTGGATGAAGAACACCGAAAGTGGCTG,
Primer set 20:
(SEQ ID No. 39)
ACGTTGGATGTGCATTGGCCACTAAAGCTC
and
(SEQ ID No. 40)
ACGTTGGATGTCGATGACTATCTGCGGATG,
Primer set 21:
(SEQ ID No. 41)
ACGTTGGATGACCCATTTGCCGAACGTGTC
and
(SEQ ID No. 42)
ACGTTGGATGTGCTTGGCGACTTTGTGCAG,
Primer set 22:
(SEQ ID No. 43)
ACGTTGGATGAGCGTGAAGAAGACGACGA
and
(SEQ ID No. 44)
ACGTTGGATGGTCTGTTGTCATTACGGGAG,
Primer set 23:
(SEQ ID No. 45)
ACGTTGGATGACATCAGGTGATGGTCATGC
and
(SEQ ID No. 46)
ACGTTGGATGCGAAGGGAACAATGGATGTG,
Primer set 24:
(SEQ ID No. 47)
ACGTTGGATGTATGCCAACCGATTTGCCTG
and
(SEQ ID No. 48)
ACGTTGGATGACATATTGTCCACCGCGTAG,
and
Primer set 25:
(SEQ ID No. 49)
ACGTTGGATGTCTTGGCAGCGGCATGGAC
and
(SEQ ID No. 50)
ACGTTGGATGCCGAATTTCCAGTCTCACAG.
2 . The primer set of claim 1 , which is applied in polymerase chain reaction to amplify a DNA fragment containing a single-nucleotide polymorphism (SNP) of M. tuberculosis.
3 . An extension primer for genotyping Mycobacterium tuberculosis selected from one of the group consisting of:
(SEQ ID No. 51)
GACCTGTCCTACGAACCGGTGATGG,
(SEQ ID No. 52)
CGTTGCCCACGTTGTTGGCG,
(SEQ ID No. 53)
CACCGGCCAACGTCTCGGGCATG,
(SEQ ID No. 54)
CCCCCGACCGGCCGTTCTTCG,
(SEQ ID No. 55)
TTCAACGGCGGCATCAT,
(SEQ ID No. 56)
GCCGAAACAAGATTTGC,
(SEQ ID No. 57)
CCTTCTGCGTCTCCAAT,
(SEQ ID No. 58)
GATATGGGGCCGCGGAT,
(SEQ ID No. 59)
ACCGTAAACGGGCCTAACCCTCC,
(SEQ ID No. 60)
TTGGGGCTGGGAACTGGG,
(SEQ ID No. 61)
ATTCACGTGAAAACCCTCG,,
(SEQ ID No. 62)
AGCTCAGCGCGCGGCTGGTGT,
(SEQ ID No. 63)
CAAAATACGGCGATCATCATGGG,
(SEQ ID No. 64)
CCACCAGTACTTGCCGC,
(SEQ ID No. 65)
ATCGGGGTGACGATGAG,
(SEQ ID No. 66)
GCCGAGGAGCCCGCGTAACCGT,
(SEQ ID No. 67)
TGTTGATCGGCCCGAGGC,
(SEQ ID No. 68)
GCGGGCGTGGAACGCTGGTC,
(SEQ ID No. 69)
AGCGTTTCCAGGTCACCGCA,
(SEQ ID No. 70)
CCAGAGCGCAACAACAA,
(SEQ ID No. 71)
CACGCTGGCATCAAGTTC,,
(SEQ ID No. 72)
GAAGACGACGAGGACGACTGGG,
(SEQ ID No. 73)
GACGATTCCGGGCATGCG,
(SEQ ID No. 74)
TGCCTGCCTGGTATGAC,
and
(SEQ ID No. 75)
GGCATGGACGGGATCGG.
4 . The extension primer of claim 3 , which is applied in polymerase chain reaction to amplify a DNA fragment having a single-nucleotide polymorphism (SNP) of M. tuberculosis as a terminal nucleotide of the DNA fragment.
5 . A combination of single-nucleotide polymorphism markers of M. tuberculosis is selected from the group consisting of “T” at position 301 of SEQ ID No.76, “A” at position 301 of SEQ ID No. 77, “A” at position 301 of SEQ ID No. 78, “G” at position 301 of SEQ ID No. 79, “G” at position 301 of SEQ ID No. 80, “G” at position 301 of SEQ ID No. 81, “C” at position 301 of SEQ ID No. 82, “G” at position 301 of SEQ ID No. 83, “C” at position 301 of SEQ ID No. 84, “A” at position 301 of SEQ ID No. 85, “A” at position 301 of SEQ ID No. 86, “A” at position 301 of SEQ ID No. 87, “G” at position 301 of SEQ ID No. 88, “A” at position 301 of SEQ ID No. 89, “G” at position 301 of SEQ ID No. 90, “G” at position 301 of SEQ ID No. 91, “A” at position 301 of SEQ ID No. 92, “C” at position 301 of SEQ ID No. 93, “C” at position 301 of SEQ ID No. 94, “T” at position 301 of SEQ ID No. 95, “T” at position 301 of SEQ ID No. 96, “T” at position 301 of SEQ ID No. 97, “T” at position 301 of SEQ ID No. 98, “T” at position 301 of SEQ ID No. 99, and “C” at position 301 of SEQ ID No.100.
6 . A method for genotyping Mycobacterium tuberculosis comprising obtaining a sample, amplifying and obtain at least one of first DNA fragment by using one or more primer sets selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50), amplifying and obtain at least one of second DNA fragment by using the obtained first DNA fragment as template and using one or more extension primers selected from the group consisting of SEQ ID Nos. 51 to 75, and detecting the second DNA fragment by using mass spectrometry.
7 . The method of claim 6 , which further comprising analyzing the mass spectrometry data based on the single-nucleotide polymorphism markers selected from the group consisting of combination of single-nucleotide polymorphism markers of M. tuberculosis is selected from the group consisting of “T” at position 301 of SEQ ID No.76, “A” at position 301 of SEQ ID No. 77, “A” at position 301 of SEQ ID No. 78, “G” at position 301 of SEQ ID No. 79, “G” at position 301 of SEQ ID No. 80, “G” at position 301 of SEQ ID No. 81, “C” at position 301 of SEQ ID No. 82, “G” at position 301 of SEQ ID No. 83, “C” at position 301 of SEQ ID No. 84, “A” at position 301 of SEQ ID No. 85, “A” at position 301 of SEQ ID No. 86, “A” at position 301 of SEQ ID No. 87, “G” at position 301 of SEQ ID No. 88, “A” at position 301 of SEQ ID No. 89, “G” at position 301 of SEQ ID No. 90, “G” at position 301 of SEQ ID No. 91, “A” at position 301 of SEQ ID No. 92, “C” at position 301 of SEQ ID No. 93, “C” at position 301 of SEQ ID No. 94, “T” at position 301 of SEQ ID No. 95, “T” at position 301 of SEQ ID No. 96, “T” at position 301 of SEQ ID No. 97, “T” at position 301 of SEQ ID No. 98, “T” at position 301 of SEQ ID No. 99, and “C” at position 301 of SEQ ID No.100.
8 . The method of claim 6 , wherein the mass spectrometry is matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
9 . The method of claim 6 , wherein the sample is bacterial culture, nasal mucus, phlegm saliva, blood, section of tissues or organ, and/or biopsy.
10 . A kit for genotyping Mycobacterium tuberculosis comprising: at least one primer set selected from the group consisting of primer sets 1 to 25 (SEQ ID Nos. 1 to 50), and at least one extension primer selected from the group consisting of SEQ ID Nos. 51 to 75.
11 . The kit of claim 10 , which further comprises a database of genotypes of M. tuberculosis based on single-nucleotide polymorphism markers.
12 . The kit of claim 11 , wherein the single-nucleotide polymorphism markers is selected from the group consisting of the combination of single-nucleotide polymorphism (SNP) markers of M. tuberculosis can be selected from the group consisting of “T” at position 301 of SEQ ID No.76, “A” at position 301 of SEQ ID No. 77, “A” at position 301 of SEQ ID No. 78, “G” at position 301 of SEQ ID No. 79, “G” at position 301 of SEQ ID No. 80, “G” at position 301 of SEQ ID No. 81, “C” at position 301 of SEQ ID No. 82, “G” at position 301 of SEQ ID No. 83, “C” at position 301 of SEQ ID No. 84, “A” at position 301 of SEQ ID No. 85, “A” at position 301 of SEQ ID No. 86, “A” at position 301 of SEQ ID No. 87, “G” at position 301 of SEQ ID No. 88, “A” at position 301 of SEQ ID No. 89, “G” at position 301 of SEQ ID No. 90, “G” at position 301 of SEQ ID No. 91, “A” at position 301 of SEQ ID No. 92, “C” at position 301 of SEQ ID No. 93, “C” at position 301 of SEQ ID No. 94, “T” at position 301 of SEQ ID No. 95, “T” at position 301 of SEQ ID No. 96, “T” at position 301 of SEQ ID No. 97, “T” at position 301 of SEQ ID No. 98, “T” at position 301 of SEQ ID No. 99, and “C” at position 301 of SEQ ID No.100.Join the waitlist — get patent alerts
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