US2018230520A1PendingUtilityA1

Primers for the detection and typing of carbapenemase-producing bacterial strains and detection method and kit

Assignee: FUNDACION PARA EL FOMENTO DE LA INVESTIG SANITARIA Y BIOMEDICA DE LA COMMUNITAT VALENCIANAPriority: Dec 31, 2012Filed: Jan 23, 2018Published: Aug 16, 2018

Est. expiryDec 31, 2032(~6.4 yrs left)· nominal 20-yr term from priority

Inventors:Gloria Royo García Juan Carlos Rodríguez Díaz Antonio José Galiana Cabrera

C12Q 2600/16C12Q 2600/156C12Q 1/689

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References

Claims

Abstract

Some pairs of primers are provided, being used for detection of carbapenemase genes and carbapenemase-producing bacterial strains, and a diagnosis method using said set of primers. Furthermore, there is also disclosed, preferably, the use of probes within each amplification reaction, more preferably multiplex PCR, for selective identification and detection of carbapenemase genes from a biological sample. The primers object of this invention may be used for preparing a kit for carbapenemase genes identification and detection, preferably carbapenemases contained in bacterial strains producing said enzymes.

Claims

exact text as granted — not AI-modified

1 - 24 . (canceled) 
     
     
         25 . A pair of primers consisting of SEQ ID NO: 52-SEQ ID NO: 53. 
     
     
         26 . A kit comprising the pair of primers according to  claim 25 , and reagents required to perform an amplification reaction. 
     
     
         27 . The kit according to  claim 26 , further comprising at least another pair of primers selected from the group consisting of SEQ ID NO: 4-SEQ ID NO: 5, SEQ ID NO: 7-SEQ ID NO: 8, SEQ ID NO: 10-SEQ ID NO: 11, SEQ ID NO: 13-SEQ ID NO: 14, SEQ ID NO: 16-SEQ ID NO: 17, SEQ ID NO: 19-SEQ ID NO: 20, SEQ ID NO: 22-SEQ ID NO: 23, SEQ ID NO: 25-SEQ ID NO: 26, SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 32, SEQ ID NO: 34-SEQ ID NO: 35, SEQ ID NO: 37-SEQ ID NO: 38, SEQ ID NO: 40-SEQ ID NO: 41, SEQ ID NO: 43-SEQ ID NO: 44, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 50, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 58-SEQ ID NO: 59, SEQ ID NO: 61-SEQ ID NO: 62, SEQ ID NO: 64-SEQ ID NO: 65, SEQ ID NO: 72-SEQ ID NO: 73, and SEQ ID NO: 75-SEQ ID NO: 76. 
     
     
         28 . The kit according to  claim 27 , further comprising at least a probe capable of hybridizing the amplification product of any of said pair of primers. 
     
     
         29 . The kit according to  claim 28 , wherein the pair of primers or the probe is labelled with a fluorophore. 
     
     
         30 . The kit according to  claim 29 , wherein the fluorophore is selected from the group consisting of cyanine 5 dye, tetramethylrhodamine, 4,5-dichloro-dimethoxy-fluorescein, and biotin. 
     
     
         31 . The kit according to  claim 28 , wherein the probe comprises the sequence SEQ ID NO: 54. 
     
     
         32 . The kit according to  claim 28 , wherein the probe comprises the sequence:
 a. SEQ ID NO: 6, when the pair of primers is SEQ ID NO: 4-SEQ ID NO: 5,   b. SEQ ID NO: 9, when the pair of primers is SEQ ID NO: 7-SEQ ID NO: 8,   c. SEQ ID NO: 12, when the pair of primers is SEQ ID NO: 10-SEQ ID NO: 11,   d. SEQ ID NO: 15, when the pair of primers is SEQ ID NO: 13-SEQ ID NO: 14,   e. SEQ ID NO: 18, when the pair of primers is SEQ ID NO: 16-SEQ ID NO: 17,   f. SEQ ID NO: 21, when the pair of primers is SEQ ID NO: 19-SEQ ID NO: 20,   g. SEQ ID NO: 24, when the pair of primers is SEQ ID NO: 22-SEQ ID NO: 23,   h. SEQ ID NO: 27, when the pair of primers is SEQ ID NO: 25-SEQ ID NO: 26,   i. SEQ ID NO: 30, when the pair of primers is SEQ ID NO: 28-SEQ ID NO: 29,   j. SEQ ID NO: 33, when the pair of primers is SEQ ID NO: 31-SEQ ID NO: 32,   k. SEQ ID NO: 36, when the pair of primers is SEQ ID NO: 34-SEQ ID NO: 35,   l. SEQ ID NO: 39, when the pair of primers is SEQ ID NO: 37-SEQ ID NO: 38,   m. SEQ ID NO: 42, when the pair of primers is SEQ ID NO: 40-SEQ ID NO: 41,   n. SEQ ID NO: 45, when the pair of primers is SEQ ID NO: 43-SEQ ID NO: 44,   o. SEQ ID NO: 48, when the pair of primers is SEQ ID NO: 46-SEQ ID NO: 47,   p. SEQ ID NO: 51, when the pair of primers is SEQ ID NO: 49-SEQ ID NO: 50,   q. SEQ ID NO: 57, when the pair of primers is SEQ ID NO: 55-SEQ ID NO: 56,   r. SEQ ID NO: 60, when the pair of primers is SEQ ID NO: 58-SEQ ID NO: 59,   s. SEQ ID NO: 63, when the pair of primers is SEQ ID NO: 61-SEQ ID NO: 62,   t. SEQ ID NO: 66, when the pair of primers is SEQ ID NO: 64-SEQ ID NO: 65,   u. SEQ ID NO: 74, when the pair of primers is SEQ ID NO: 72-SEQ ID NO: 73, or   v. SEQ ID NO: 77, when the pair of primers is SEQ ID NO: 75-SEQ ID NO: 76.   
     
     
         33 . The kit according to  claim 27 , comprising the pairs of primers SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 55-SEQ ID NO: 56, and SEQ ID NO: 72-SEQ ID NO: 73. 
     
     
         34 . An in vitro method for detection and/or typing of SPM carbapenemase-producing bacterial strains in an isolated biological sample, said method comprising:
 i. contacting an isolated biological sample comprising DNA with the pair of primers according to  claim 25 ,   ii. amplifying the DNA comprised in said isolated biological sample with the pair of primers, and   iii. detecting the presence of the amplification product obtained in step (ii), wherein the presence of said amplification product is indicative of the presence of SPM carbapenemase-producing bacterial strains in the isolated biological sample.   
     
     
         35 . The in vitro method according to  claim 34 , wherein step (i) additionally comprises contacting the isolated biological sample with a further pair of primers selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 2, SEQ ID NO: 4-SEQ ID NO: 5, SEQ ID NO: 7-SEQ ID NO: 8, SEQ ID NO: 10-SEQ ID NO: 11, SEQ ID NO: 13-SEQ ID NO: 14, SEQ ID NO: 16-SEQ ID NO: 17, SEQ ID NO: 19-SEQ ID NO: 20, SEQ ID NO: 22-SEQ ID NO: 23, SEQ ID NO: 25-SEQ ID NO: 26, SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 32, SEQ ID NO: 34-SEQ ID NO: 35, SEQ ID NO: 37-SEQ ID NO: 38, SEQ ID NO: 40-SEQ ID NO: 41, SEQ ID NO: 43-SEQ ID NO: 44, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 50, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 58-SEQ ID NO: 59, SEQ ID NO: 61-SEQ ID NO: 62, SEQ ID NO: 64-SEQ ID NO: 65, SEQ ID NO: 67-SEQ ID NO: 68, SEQ ID NO: 72-SEQ ID NO: 73, and SEQ ID NO: 75-SEQ ID NO: 76. 
     
     
         36 . The in vitro method according to  claim 34 , wherein detection of step (iii) comprises hybridization of the amplification product obtained in step (ii) with at least a probe capable of hybridizing with the amplification product of the pair of primers used in step (i). 
     
     
         37 . The in vitro method according to  claim 36 , wherein the pair of primers or the probe is labelled with a fluorophore. 
     
     
         38 . The in vitro method according to  claim 37 , wherein the fluorophore is selected from the group consisting of cyanine 5 dye, tetramethylrhodamine, 4,5-dichloro-dimethoxy-fluorescein, and biotin. 
     
     
         39 . The in vitro method according to  claim 36 , wherein the probe comprises the sequence SEQ ID NO: 54. 
     
     
         40 . The in vitro method according to  claim 36 , wherein the probe comprises the sequence:
 a. SEQ ID No: 3, when the pair of primers is SEQ ID No: 1-SEQ ID No: 2,   b. SEQ ID NO: 6, when the pair of primers is SEQ ID NO: 4-SEQ ID NO: 5,   c. SEQ ID NO: 9, when the pair of primers is SEQ ID NO: 7-SEQ ID NO: 8,   d. SEQ ID NO: 12, when the pair of primers is SEQ ID NO: 10-SEQ ID NO: 11,   e. SEQ ID NO: 15, when the pair of primers is SEQ ID NO: 13-SEQ ID NO: 14,   f. SEQ ID NO: 18, when the pair of primers is SEQ ID NO: 16-SEQ ID NO: 17,   g. SEQ ID NO: 21, when the pair of primers is SEQ ID NO: 19-SEQ ID NO: 20,   h. SEQ ID NO: 24, when the pair of primers is SEQ ID NO: 22-SEQ ID NO: 23,   i. SEQ ID NO: 27, when the pair of primers is SEQ ID NO: 25-SEQ ID NO: 26,   j. SEQ ID NO: 30, when the pair of primers is SEQ ID NO: 28-SEQ ID NO: 29,   k. SEQ ID NO: 33, when the pair of primers is SEQ ID NO: 31-SEQ ID NO: 32,   l. SEQ ID NO: 36, when the pair of primers is SEQ ID NO: 34-SEQ ID NO: 35,   m. SEQ ID NO: 39, when the pair of primers is SEQ ID NO: 37-SEQ ID NO: 38,   n. SEQ ID NO: 42, when the pair of primers is SEQ ID NO: 40-SEQ ID NO: 41,   o. SEQ ID NO: 45, when the pair of primers is SEQ ID NO: 43-SEQ ID NO: 44,   p. SEQ ID NO: 48, when the pair of primers is SEQ ID NO: 46-SEQ ID NO: 47,   q. SEQ ID NO: 51, when the pair of primers is SEQ ID NO: 49-SEQ ID NO: 50,   r. SEQ ID NO: 57, when the pair of primers is SEQ ID NO: 55-SEQ ID NO: 56,   s. SEQ ID NO: 60, when the pair of primers is SEQ ID NO: 58-SEQ ID NO: 59,   t. SEQ ID NO: 63, when the pair of primers is SEQ ID NO: 61-SEQ ID NO: 62,   u. SEQ ID NO: 66, when the pair of primers is SEQ ID NO: 64-SEQ ID NO: 65,   v. SEQ ID No: 69, SEQ ID No: 70 and/or SEQ ID No: 71, when the pair of primers is SEQ ID No: 67-SEQ ID No: 68,   w. SEQ ID NO: 74, when the pair of primers is SEQ ID NO: 72-SEQ ID NO: 73, or   x. SEQ ID NO: 77, when the pair of primers is SEQ ID NO: 75-SEQ ID NO: 76.   
     
     
         41 . The in vitro method according to  claim 35 , comprising in step (i) contacting an isolated biological sample with all the pairs of primers: SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 67-SEQ ID NO: 68 and SEQ ID NO: 72-SEQ ID NO: 73. 
     
     
         42 . The in vitro method according to  claim 35 , comprising in step (i) contacting an isolated biological sample with all the pairs of primers SEQ ID NO: 1-SEQ ID NO: 2, SEQ ID NO: 4-SEQ ID NO: 5, SEQ ID NO: 7-SEQ ID NO: 8, SEQ ID NO: 10-SEQ ID NO: 11, SEQ ID NO: 13-SEQ ID NO: 14, SEQ ID NO: 16-SEQ ID NO: 17, SEQ ID NO: 19-SEQ ID NO: 20, SEQ ID NO: 22-SEQ ID NO: 23, SEQ ID NO: 25-SEQ ID NO: 26, SEQ ID NO: 28-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 32, SEQ ID NO: 34-SEQ ID NO: 35, SEQ ID NO: 37-SEQ ID NO: 38, SEQ ID NO: 40-SEQ ID NO: 41, SEQ ID NO: 43-SEQ ID NO: 44, SEQ ID NO: 46-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 50, SEQ ID NO: 52-SEQ ID NO: 53, SEQ ID NO: 55-SEQ ID NO: 56, SEQ ID NO: 58-SEQ ID NO: 59, SEQ ID NO: 61-SEQ ID NO: 62, SEQ ID NO: 64-SEQ ID NO: 65, SEQ ID NO:67-SEQ ID NO:68, SEQ ID NO: 72-SEQ ID NO: 73 and SEQ ID NO: 75-SEQ ID NO: 76. 
     
     
         43 . The in vitro method according to  claim 34 , wherein the amplification step (ii) is carried out by means of PCR. 
     
     
         44 . The in vitro method according to  claim 34 , wherein the biological sample comes from an individual. 
     
     
         45 . The in vitro method according to  claim 44 , wherein the individual is a human. 
     
     
         46 . The in vitro method according to  claim 44 , wherein the biological sample is blood, sputum, exudate, skin or hemoculture previously inoculated with blood from an individual preferably having bacteraemia. 
     
     
         47 . The method according to  claim 34 , further comprising:
 (iv) assigning the individual to the group of patients infected with carbapenemase-producing bacterial strains when in step (iii) the presence of the amplification product is detected.

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