Primers and kits for colony multiplex pcr for the detection of class a, b, c, and d beta-lactamase genes and methods of using thereof

Abstract

The present invention provides kits and primers for colony multiplex PCR for the detection of class A, B, C, and D β-lactamase genes. The rapid detection of bla genes by using the kits and primers according to the present invention allows appropriate prescribing of antibiotics, which can reduce patient mortality and minimize antibiotic resistance. The present invention provides kits and primers for a rapid and accurate molecular method to overcome (a) to detect all clinically-important bla genes and (b) to explain phenotypic tests' results well by using 54 primer pairs, which are designed through novel and elaborate optimization processes. With perfect specificity and sensitivity in 172 control strains and 403 clinical strains, the present invention provides prompt and clinical application to the identification of all bla genes in bacterial pathogens.

What is claimed is: 
     
         1 . A primer pair selected from the primer pair group consisting of a pair of Seq. No. 1 and 2, a pair of Seq. 3 and 4, a pair of Seq. No. 5 and 6, a pair of Seq. No. 7 and 8, a pair of Seq. No. 9 and 10, a pair of Seq. No. 11 and 12, a pair of Seq. No. 13 and 14, a pair of Seq. No. 15 and 16, a pair of Seq. No. 17 and 18, a pair of Seq. No. 19 and 20, a pair of Seq. No. 21 and 22, a pair of Seq. No. 23 and 24, a pair of Seq. No. 25 and 26, a pair of Seq. No. 27 and 28, a pair of Seq. No. 29 and 30, a pair of Seq. No. 31 and 32, a pair of Seq. No. 33 and 34, a pair of Seq. No. 35 and 36, a pair of Seq. No. 37 and 38, a pair of Seq. No. 39 and 40, a pair of Seq. No. 41 and 42, a pair of Seq. No. 43 and 44, a pair of Seq. No. 45 and 46, a pair of Seq. No. 47 and 48, a pair of Seq. No. 49 and 50, a pair of Seq. No. 51 and 52, a pair of Seq. No. 53 and 54, No. 55 and 56, a pair of Seq. 57 and 58, a pair of Seq. No. 59 and 60, a pair of Seq. No. 61 and 62, a pair of Seq. No. 63 and 64, a pair of Seq. No. 65 and 66, a pair of Seq. No. 67 and 68, a pair of Seq. No. 69 and 70, a pair of Seq. No. 71 and 72, a pair of Seq. No. 73 and 74, a pair of Seq. No. 75 and 76, a pair of Seq. No. 77 and 78, a pair of Seq. No. 79 and 80, a pair of Seq. No. 81 and 82, a pair of Seq. No. 83 and 84, a pair of Seq. No. 85 and 86, a pair of Seq. No. 87 and 88, a pair of Seq. No. 89 and 90, a pair of Seq. No. 91 and 92, a pair of Seq. No. 93 and 94, a pair of Seq. No. 95 and 96, a pair of Seq. No. 97 and 98, a pair of Seq. No. 99 and 100, a pair of Seq. No. 101 and 102, a pair of Seq. No. 103 and 104, a pair of Seq. No. 105 and 106, and a pair of Seq. No. 107 and 108; and each of full-length complement primer pairs thereof for identifying β-lactamase nucleic acid. 
     
     
         2 . Primer pairs having at least 2 primer pairs selected from the group consisting of a pair of Seq. No. 1 and 2, a pair of Seq. 3 and 4, a pair of Seq. No. 5 and 6, a pair of Seq. No. 7 and 8, a pair of Seq. No. 9 and 10, a pair of Seq. No. 11 and 12, a pair of Seq. No. 13 and 14, a pair of Seq. No. 15 and 16, a pair of Seq. No. 17 and 18, a pair of Seq. No. 19 and 20, a pair of Seq. No. 21 and 22, a pair of Seq. No. 23 and 24, a pair of Seq. No. 25 and 26, a pair of Seq. No. 27 and 28, a pair of Seq. No. 29 and 30, a pair of Seq. No. 31 and 32, a pair of Seq. No. 33 and 34, a pair of Seq. No. 35 and 36, a pair of Seq. No. 37 and 38, a pair of Seq. No. 39 and 40, a pair of Seq. No. 41 and 42, a pair of Seq. No. 43 and 44, a pair of Seq. No. 45 and 46, a pair of Seq. No. 47 and 48, a pair of Seq. No. 49 and 50, a pair of Seq. No. 51 and 52, a pair of Seq. No. 53 and 54, No. 55 and 56, a pair of Seq. 57 and 58, a pair of Seq. No. 59 and 60, a pair of Seq. No. 61 and 62, a pair of Seq. No. 63 and 64, a pair of Seq. No. 65 and 66, a pair of Seq. No. 67 and 68, a pair of Seq. No. 69 and 70, a pair of Seq. No. 71 and 72, a pair of Seq. No. 73 and 74, a pair of Seq. No. 75 and 76, a pair of Seq. No. 77 and 78, a pair of Seq. No. 79 and 80, a pair of Seq. No. 81 and 82, a pair of Seq. No. 83 and 84, a pair of Seq. No. 85 and 86, a pair of Seq. No. 87 and 88, a pair of Seq. No. 89 and 90, a pair of Seq. No. 91 and 92, a pair of Seq. No. 93 and 94, a pair of Seq. No. 95 and 96, a pair of Seq. No. 97 and 98, a pair of Seq. No. 99 and 100, a pair of Seq. No. 101 and 102, a pair of Seq. No. 103 and 104, a pair of Seq. No. 105 and 106, and a pair of Seq. No. 107 and 108; and each of full-length complement primer pairs thereof for identifying β-lactamase nucleic acid. 
     
     
         3 . Primer pairs according to  claim 2 , having at least 5 primer pairs selected from the group consisting of a pair of Seq. No. 1 and 2, a pair of Seq. 3 and 4, a pair of Seq. No. 5 and 6, a pair of Seq. No. 7 and 8, a pair of Seq. No. 9 and 10, a pair of Seq. No. 11 and 12, a pair of Seq. No. 13 and 14, a pair of Seq. No. 15 and 16, a pair of Seq. No. 17 and 18, a pair of Seq. No. 19 and 20, a pair of Seq. No. 21 and 22, a pair of Seq. No. 23 and 24, a pair of Seq. No. 25 and 26, a pair of Seq. No. 27 and 28, a pair of Seq. No. 29 and 30, a pair of Seq. No. 31 and 32, a pair of Seq. No. 33 and 34, a pair of Seq. No. 35 and 36, a pair of Seq. No. 37 and 38, a pair of Seq. No. 39 and 40, a pair of Seq. No. 41 and 42, a pair of Seq. No. 43 and 44, a pair of Seq. No. 45 and 46, a pair of Seq. No. 47 and 48, a pair of Seq. No. 49 and 50, a pair of Seq. No. 51 and 52, a pair of Seq. No. 53 and 54, No. 55 and 56, a pair of Seq. 57 and 58, a pair of Seq. No. 59 and 60, a pair of Seq. No. 61 and 62, a pair of Seq. No. 63 and 64, a pair of Seq. No. 65 and 66, a pair of Seq. No. 67 and 68, a pair of Seq. No. 69 and 70, a pair of Seq. No. 71 and 72, a pair of Seq. No. 73 and 74, a pair of Seq. No. 75 and 76, a pair of Seq. No. 77 and 78, a pair of Seq. No. 79 and 80, a pair of Seq. No. 81 and 82, a pair of Seq. No. 83 and 84, a pair of Seq. No. 85 and 86, a pair of Seq. No. 87 and 88, a pair of Seq. No. 89 and 90, a pair of Seq. No. 91 and 92, a pair of Seq. No. 93 and 94, a pair of Seq. No. 95 and 96, a pair of Seq. No. 97 and 98, a pair of Seq. No. 99 and 100, a pair of Seq. No. 101 and 102, a pair of Seq. No. 103 and 104, a pair of Seq. No. 105 and 106, and a pair of Seq. No. 107 and 108; and each of full-length complement primer pairs thereof for identifying β-lactamase nucleic acid. 
     
     
         4 . Primer pairs according to  claim 3 , having at least 8 primer pairs selected from the group consisting of a pair of Seq. No. 1 and 2, a pair of Seq. 3 and 4, a pair of Seq. No. 5 and 6, a pair of Seq. No. 7 and 8, a pair of Seq. No. 9 and 10, a pair of Seq. No. 11 and 12, a pair of Seq. No. 13 and 14, a pair of Seq. No. 15 and 16, a pair of Seq. No. 17 and 18, a pair of Seq. No. 19 and 20, a pair of Seq. No. 21 and 22, a pair of Seq. No. 23 and 24, a pair of Seq. No. 25 and 26, a pair of Seq. No. 27 and 28, a pair of Seq. No. 29 and 30, a pair of Seq. No. 31 and 32, a pair of Seq. No. 33 and 34, a pair of Seq. No. 35 and 36, a pair of Seq. No. 37 and 38, a pair of Seq. No. 39 and 40, a pair of Seq. No. 41 and 42, a pair of Seq. No. 43 and 44, a pair of Seq. No. 45 and 46, a pair of Seq. No. 47 and 48, a pair of Seq. No. 49 and 50, a pair of Seq. No. 51 and 52, a pair of Seq. No. 53 and 54, No. 55 and 56, a pair of Seq. 57 and 58, a pair of Seq. No. 59 and 60, a pair of Seq. No. 61 and 62, a pair of Seq. No. 63 and 64, a pair of Seq. No. 65 and 66, a pair of Seq. No. 67 and 68, a pair of Seq. No. 69 and 70, a pair of Seq. No. 71 and 72, a pair of Seq. No. 73 and 74, a pair of Seq. No. 75 and 76, a pair of Seq. No. 77 and 78, a pair of Seq. No. 79 and 80, a pair of Seq. No. 81 and 82, a pair of Seq. No. 83 and 84, a pair of Seq. No. 85 and 86, a pair of Seq. No. 87 and 88, a pair of Seq. No. 89 and 90, a pair of Seq. No. 91 and 92, a pair of Seq. No. 93 and 94, a pair of Seq. No. 95 and 96, a pair of Seq. No. 97 and 98, a pair of Seq. No. 99 and 100, a pair of Seq. No. 101 and 102, a pair of Seq. No. 103 and 104, a pair of Seq. No. 105 and 106, and a pair of Seq. No. 107 and 108; and each of full-length complement primer pairs thereof for identifying β-lactamase nucleic acid. 
     
     
         5 . Primer pairs according to  claim 4 , consisting of a pair of Seq. No. 1 and 2, a pair of Seq. 3 and 4, a pair of Seq. No. 5 and 6, a pair of Seq. No. 7 and 8, a pair of Seq. No. 9 and 10, a pair of Seq. No. 11 and 12, a pair of Seq. No. 13 and 14, a pair of Seq. No. 15 and 16, a pair of Seq. No. 17 and 18, a pair of Seq. No. 19 and 20, a pair of Seq. No. 21 and 22, a pair of Seq. No. 23 and 24, a pair of Seq. No. 25 and 26, a pair of Seq. No. 27 and 28, a pair of Seq. No. 29 and 30, a pair of Seq. No. 31 and 32, a pair of Seq. No. 33 and 34, a pair of Seq. No. 35 and 36, a pair of Seq. No. 37 and 38, a pair of Seq. No. 39 and 40, a pair of Seq. No. 41 and 42, a pair of Seq. No. 43 and 44, a pair of Seq. No. 45 and 46, a pair of Seq. No. 47 and 48, a pair of Seq. No. 49 and 50, a pair of Seq. No. 51 and 52, a pair of Seq. No. 53 and 54, No. 55 and 56, a pair of Seq. 57 and 58, a pair of Seq. No. 59 and 60, a pair of Seq. No. 61 and 62, a pair of Seq. No. 63 and 64, a pair of Seq. No. 65 and 66, a pair of Seq. No. 67 and 68, a pair of Seq. No. 69 and 70, a pair of Seq. No. 71 and 72, a pair of Seq. No. 73 and 74, a pair of Seq. No. 75 and 76, a pair of Seq. No. 77 and 78, a pair of Seq. No. 79 and 80, a pair of Seq. No. 81 and 82, a pair of Seq. No. 83 and 84, a pair of Seq. No. 85 and 86, a pair of Seq. No. 87 and 88, a pair of Seq. No. 89 and 90, a pair of Seq. No. 91 and 92, a pair of Seq. No. 93 and 94, a pair of Seq. No. 95 and 96, a pair of Seq. No. 97 and 98, a pair of Seq. No. 99 and 100, a pair of Seq. No. 101 and 102, a pair of Seq. No. 103 and 104, a pair of Seq. No. 105 and 106, and a pair of Seq. No. 107 and 108; and each of full-length complement primer pairs thereof for identifying β-lactamase nucleic acid. 
     
     
         6 . A primer pair selected from the primer pair group consisting of a pair of Seq. No. 1 and 2, a pair of Seq. 3 and 4, a pair of Seq. No. 5 and 6, a pair of Seq. No. 7 and 8, a pair of Seq. No. 9 and 10, a pair of Seq. No. 11 and 12, a pair of Seq. No. 13 and 14, a pair of Seq. No. 15 and 16, a pair of Seq. No. 17 and 18, a pair of Seq. No. 19 and 20, a pair of Seq. No. 21 and 22, a pair of Seq. No. 23 and 24, and a pair of Seq. No. 25 and 26; and each of full-length complement primer pairs thereof for identifying Class A β-lactamase nucleic acid.