Resumen
The present invention relates to a method for the detection and/or quantification of Streptococcus pneumoniae, a Gram-positive bacteria being an important human pathogen, in an isolated biological sample, through magneto-amperometric biosensors, comprising detecting fragments of the lytA gene of said microorganism, amplified through PCR, preferably asymmetric PCR or direct asymmetric PCR, by means of the hybridisation thereof to a specific probe fully complementary to a region of said amplified fragment. Furthermore, the present invention additionally refers to the use of primers SEQ ID NO: 3 and 4 together with probes SEQ ID NO: 1 and/or SEQ ID NO: 2 for the purpose of carrying out said detection. The biosensor developed to detect S . pneumoniae renders it applicable to different types of clinical samples proceeding from patients infected with this bacteria or other related bacteria. Preferably the clinical sample is an isolated biological fluid such as blood, cephalorachidian liquid, saliva or urine.
Reivindicaciones
1. Method to detect and/or quantifyStreptococcus pneumoniae in at least one isolated biological sample, through magneto-amperometric biosensors, comprising: a. immobilizing on a solid support a suspension of superparamagnetic particles (MBs) labeled with a compound A and with the probe of DNA SEQ ID NO: 1 labeled on its 5' end with a compound B related to compound A, wherein said particles and probe are bound by the binding of compounds A-B, b. adding to the mixture of step (a) the product of the amplification through PCR obtained by using the direct primer SEQ ID NO: 3 and the reverse primer SEQ ID NO: 4 of thelytA gene of S. pneumoniae, from an isolated biological sample, wherein the primer SEQ ID NO: 4 is bound by its 5' end to compound B, and c. detecting the hybridization of the probe SEQ ID NO: 1 of step (a) to the DNA fragment amplified in step (b), by adding the compound A conjugated to an element capable of being detected and/or quantified. 2. Method according to claim 1, wherein the biological sample is a biological fluid. 3. Method according to claim 2, wherein the isolated biological fluid is blood, cerebrospinal fluid, saliva or urine. 4. Method according to any of the claims 1 to 3, wherein PCR is asymmetric. 5. Method according to claim 4, where, in said asymmetric PCR an amount of the primer SEQ ID NO: 3 is used that is 8 to 12 times smaller than SEQ ID NO: 4 bound by its 5' end to compound B. 6. Method according to any of the claims 4 or 5, wherein the asymmetric PCR is direct. 7. Method according to any of the claims 1 to 6, wherein compound A conjugated to an element capable of being detected and/or quantified according to step (c) is, in turn, covalently conjugated to a hydrophilic polymer backbone. 8. Method according to any of the claims 1 to 7, wherein compound A is streptavidin and compound B is biotin. 9. Method according to any of the claims 1 to 8, wherein the element capable of being detected and/or quantified according to step (c) is an oxidase enzyme. 10. Method according to claim 9, wherein the oxidase enzyme is a peroxidase and said detection and/or quantification is carried out by adding H<2> O<2> . 11. Method according to any of the claims 1 to 10 for monitoring the response to aS .pneumoniae treatment. 12. Use of the direct primer SEQ ID NO: 3 and the reverse primer SEQ ID NO: 4 for the amplification through PCR of a DNA fragment oflytA gene ofS .pneumoniae and its detection and/or quantification with the probe SEQ ID NO: 1 and/or with the probe SEQ ID NO: 2, wherein said probes have a labeling that allows an isotopic, colorimetric, fluorometric or electrochemical detection, using as a template the DNA present in an isolated biological sample. 13. Use according to claim 12, wherein said probes have a labeling that allows an electrochemical detection. 14. Use according to any of the claims 12 or 13, wherein the isolated biological sample is an isolated biological fluid. 15. Use according to claim 14, wherein the isolated biological fluid is blood, cerebrospinal fluid, saliva or urine. 16. Use according to any of the claims 12 to 15, wherein PCR is asymmetric. 17. Use according to claim 16, wherein the asymmetric PCR is direct. 18. Use according to any of the claims 12 to 17 for monitoring the response to aS .pneumoniae treatment. 19. Kit comprising the direct primer SEQ ID NO: 3, the reverse primer SEQ ID NO: 4, and the probe SEQ ID NO: 1 and/or the probe SEQ ID NO: 2, wherein said probes have a labeling that allows an isotopic, colorimetric, fluorometric or electrochemical detection. 20. Use of the kit according to claim 19 for the detection and/or quantification ofS .pneumoniae. 21. Use of the kit according to claim 19 for monitoring the response to aS .pneumoniae treatment.
