One-tube nested PCR for detecting mycobacterium tuberculosis

A method for detecting the presence or absence of Mycobacterium tuberculosis in a sample using a nested polymerase chain reaction, comprising

• (i) adding into one tube with DNA from the sample, buffer, dNTP, Taq polymerase, probe having SEQ ID NO:
• 10, an outer pair of oligonucleotide primers selected from the group consisting of (a) SEQ ID NOS:
• 2 and 4, and (b) SEQ ID NOS:
• 3 and 5, and an inner pair of oligonucleotide primers selected from the group consisting of (c) SEQ ID NOS:
• 6 and 8, and (d) SEQ ID NOS:
• 7 and 9 to form a mixture, wherein the concentration of the outer pair of oligonucleotide primers to the concentration of the inner pair of oligonucleotide primers is 1:5 o 1:100;
• (ii) performing first 20-50 cycles of amplification for PCR products from the outer primers in the first-stage polymerase chain reaction;
• (iii) performing following 10-30 cycles of amplification PCR products from for the inner primers in the second-stage polymerase chain reaction;;
• and
• (iv) identifying M. tuberculosis by the probe.

A nucleotide sequence for detecting the presence or absence of M. tuberculosis is selected from the group consisting of

• 5′-CACGTAGGCG AACCCTGCCC AGGTC-3′
• SEQ ID NO:2
• 5′-GCGTAGGCGT CGGTCACAAA-3′
• SEQ ID NO:3
• 5′-GTGAGGGCAT CGAGGTGGCC AGATG-3′
• SEQ ID NO:4
• 5′-CGTGAGGGCA TCGAGGTGGC-3′
• SEQ ID NO:5
• 5′-GACACATAGG TGAGGTCTGC-3′
• SEQ ID NO:6
• 5′-GCCACGTAGG CGAACCCTG-3′
• SEQ ID NO:7
• 5′-ACGGCTGA TGACCAAACT-3′
• SEQ ID NO:8
• 5′-AGATGCACCG TCGAACGG-3′
• SEQ ID NO:9
• TTTTTTTTTTACCTAACCGGCTGTGGGTAGCAGA.
• SEQ ID NO:10.