Resumen
A simple method for the collection of field samples for nucleic acid extraction and subsequential detection of microorganisms from various media such as aqueous samples, sediments and biofilms (sessile bacteria). This method is a quantitative polymerase chain reaction (PCR)-based technique for detection of SRP in various media (i.e., aqueous samples, sediments, biofilms). The principle of this technique is based on the fact that all prokaryotes (Bacteria and Archaea) able to carry out sulfate reduction possess a gene that encodes dissimilatory sulfite reductase (DSR), the key enzyme in the sulfate reduction pathway. Microbial induced corrosion is due to bacterial growth and thus, tests for the detection of bacterial growth in fluids involved in oil processing are routinely performed. Thus far, the widely accepted methods for detection of bacterial growth of microorganisms involved in corrosion are cultivation dependent and, therefore, these methods underestimate the number of microorganisms present in a sample and require long incubation times.
Reivindicaciones
WHAT IS CLAIMED IS: 1. A method of detecting the presence of an organism in an industrial water, comprising: adding a sample of the industrial water to a buffer to give a buffered sample; isolating a nucleic acid from the organism, if any, in the buffered sample to give an isolated nucleic acid; amplifying a nucleic acid sequence associated with the organism from the isolated nucleic acid to give an amplified nucleic acid; and wherein an observance of the amplified nucleic acid indicates the presence of the organism in the industrial water. 2. The method of claim 1, wherein the organism in the industrial water is a sulfate reducing bacteria. 3. The method of claim 1, wherein the nucleic acid sequence associated with the organism in the industrial water is amplified using a polymerase chain reaction. 4. The method of claim 3, wherein the polymerase chain reaction is carried out using a first and a second primer. 5. The method of claim 1, wherein the amplification of the nucleic acid sequence associated with the organism in the industrial water is monitored using a binding probe, wherein the binding probe binds to a portion of the nucleic acid sequence associated with the organism in the industrial water. 6. The method of claim 5, wherein binding of the binding probe to the portion of the nucleic acid sequence is used to quantitate the amplified nucleic acid in order to indicate the amount of organism in the industrial water. 7. The method of claim 4, wherein the first primer and second primer are DsrUnivlFM and DsrUniv225R, DsrUniv43F and DsrUniv225R, DsrUnivl577F and DsrUnivl712R, DsrUnivlFM and DsrUniv43R, or DsrUnivl712F and DsrUniv4R, respectively. 8. The method of claim 5, wherein the binding probe comprises SEQ ID NO:6 or a portion thereof, or SEQ ID NO: 7 or a portion thereof. 9. The method of claim 1 , further comprising the step of filtering the sample of the industrial water through a filter. 10. The method of claim 9, further comprising the step of shredding the filter before isolating nucleic acids from the sample. 11. The method of claim 9, wherein the filter comprises polyvinylidene fluoride (PVDF). 12. The method of claim 9, wherein the filter has a porosity of approximately O.l[mu]m. 13. The method of claim 1, further comprising the step of transporting the sample of the industrial water to a laboratory facility. 14. The method of claim 13, wherein the sample of the industrial water is transported at a temperature of about O<0>C to 22<0>C. 15. The method of claim 13, wherein the sample of the industrial water is transported at below about 22<0>C. 16. The method of claim 13 wherein the sample of the industrial water is transported to the laboratory facility within about 1 week of having been collected. 17. The method of claim 13, wherein the sample of the industrial water is transported to the laboratory facility within about 2 days of having been collected. 18. A method of detecting the presence of an organism in an industrial water, comprising: filtering a sample of the industrial water through a filter to give a material on the filter; adding a buffer to the material on the filter; isolating a nucleic acid from the organism, if any, from the material on the filter to give an isolated nucleic acid; amplifying a nucleic acid sequence associated with the organism from the isolated nucleic acid to give an amplified nucleic acid; and wherein an occurrence of the amplified nucleic acid indicates the presence of the organism in the industrial water. 19. The method of claim 18, wherein the organism in the industrial water is a sulfate reducing bacteria. 20. A method for isolating an organism in an industrial water comprising: filtering a sample of the industrial water through a filter to give the organism on the filter. 21. The method of claim 20, wherein the filter has a porosity of approximately 0.1 [mu]m. 22. The method of claim 20 further comprising adding a buffer to the organism on the filter to give a buffer mixture. 23. The method of claim 20 further comprising transporting the buffer mixture to a different location. 24. A method of detecting the presence of sulfate reducing bacteria in an industrial water, comprising: filtering a sample of the industrial water through a filter to give a material on the filter; adding a buffer to the material on the filter; isolating a nucleic acid from the sulfate reducing bateria, if any, from the material on the filter to give an isolated nucleic acid; amplifying a nucleic acid sequence comprising a sequence from a dsrA or dsrB gene from the isolated nucleic acid to give an amplified nucleic acid; and wherein an occurrence of the amplified nucleic acid indicates the presence of the sulfate reducing bacteria in the industrial water. 25. A method of detecting the presence of sulfate reducing prokaryotes in a sample which may contain said prokaryotes, comprising: obtaining a sample susceptible of containing said prokaryote; isolating nucleic acid from said sample; exposing nucleic acid to a binding probe, wherein said binding probe binds to a portion of the dsr gene found in sulfate reducing prokaryotes; exposing said nucleic acid to a first and second primer, amplifying said exposed nucleic acid by PCR amplification; and quantifying the PCR amplified nucleic acid by detection of said detector molecule. 26. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers DsrUniv43F and DsrUniv225R wherein the primer DsrUniv43F comprises SEQ E) NO:4 or a portion thereof and the primer DsrUniv225F comprises SEQ E) NO: 5 or a portion thereof. 27. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers DsrUnivl577F and DsrUniv 1712R wherein the primer DsrUniv 1577F comprises SEQ ED NO : 1 or a portion thereof and the primer DsrUnivl712F comprises SEQ ID NO: 2 or a portion thereof. 28. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers Dsr IFM and DsrUniv225R wherein the primer DsrlFM comprises SEQ E) NO:3 or a portion thereof and the primer DsrUniv225F comprises SEQ E) NO:5 or a portion thereof. 29. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers DsrUniv IFM and DsrUniv43R wherein the primer DsrUnivlFM comprises SEQ E) NO:3 or a portion thereof and the primer DsrUniv43R comprises SEQ E) NO: 8 or a portion thereof. 30. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers Dsrl712F and DsrUniv4RM wherein the primer Dsrl712F comprises SEQ E) NO:9 or a portion thereof and the primer DsrUniv4RM comprises SEQ E) NO: 10 or a portion thereof. 31. A PCR probe composition capable of identifying sulfate reducing prokaryotes, the composition comprising the probes DsrUnivPF wherein the probe DsrUnivPF comprises SEQ E) NO:6 or a portion thereof. 32. A PCR probe composition capable of identifying sulfate reducing prokaryotes, the composition comprising the probes DsrUnivPR wherein the probe DsrUnivPR comprises SEQ ID NO: 7 or a portion thereof.
