WO2009016652A1 - A buffer system and a method for direct pcr amplification - Google Patents

A buffer system and a method for direct pcr amplification Download PDF

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Publication number
WO2009016652A1
WO2009016652A1 PCT/IN2008/000477 IN2008000477W WO2009016652A1 WO 2009016652 A1 WO2009016652 A1 WO 2009016652A1 IN 2008000477 W IN2008000477 W IN 2008000477W WO 2009016652 A1 WO2009016652 A1 WO 2009016652A1
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Prior art keywords
buffer system
blood
pcr
sample
buffer
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PCT/IN2008/000477
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French (fr)
Inventor
Jagannath Manjula
Chandrasekhar Bhaskaran Nair
Pillarisetti Venkata Subbarao
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Bigtec Private Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • This invention relates to the PCR amplification and detection of nucleic acids from blood, plasma, serum or any other whole microbial cells with out nucleic acid isolation by employing a unique buffer formulation.
  • This system can be used for the detection of viruses such as Cytomegalovirus. Herpes simplex virus, Epstein-Ban- virus, Hepatitis B. or Hepatitis C virus and any blood-borne bacteria.
  • the present invention is related to direct PCR with blood, plasma, serum or any other whole microbial cells using a unique buffer formulation without the need for nucleic acid isolation.
  • the buffer mentioned here is a universal buffer for any type of blood sample. This will find wide applications in diagnosis of clinical pathogens and also for direct PCRs from whole microbial cells.
  • Blood samples are extensively used for PCR-based diagnosis of microbial infections, genetic diseases, forensic analysis, as well as for blood banking.
  • Inhibitors in blood which have been identified, are either natural components of blood, mainly heme and leukocyte DNA, or added anticoagulants such as EDTA and heparin.
  • immunoglobulin G present in human plasma was identified as a major inhibitor of diagnostic PCR in blood.
  • Widely used thermostable polymerases like Thermus aquaticus DNA polymerase (Taq) and AmpliTaq Gold are completely inhibited in the presence of 0.004-0.2% whole human blood.
  • United States Patent no 5501963 reports the use of different types of buffers depending on the concentration of the monovalent and bivalent ions present in the blood sample to achieve the PCR reaction. As an additional step to ensure efficient amplification the US patent 5501963 employs few cycles of heating and cooling before carrying out the actual PCR reaction.
  • the main object of the present invention is to obtain a buffer system for direct PCR amplification.
  • Another main object of the present invention is to develop a method for direct PCR amplification of samples.
  • the present invention relates to a buffer system for direct PCR amplification
  • said buffer system comprises a buffer salt, a chloride or sulphate containing bivalent ion, a non-ionic detergent, a stabilizer and a sugar alcohol optionally alongwith a chloride containing monovalent ion and a method for direct PCR amplification of samples, wherein the method comprises steps of: (a) adding buffer system of claim 1, suitable primers, dNTP's and polymerase to the sample to obtain a PCR mixture; and (b) subjecting the PCR mixture to predetermined conditions for the amplification of nucleic acid in the sample.
  • the present invention relates to a buffer system for direct PCR amplification, said buffer system comprises a buffer salt, a chloride or sulphate containing bivalent ion, a non-ionic detergent, a stabilizer and a sugar alcohol optionally alongwith a chloride containing monovalent ion.
  • said buffer system is used preferably to detect infections.
  • said samples are selected from a group comprising blood, plasma, serum and whole microbial cells.
  • said buffer is Tris-HCl at a concentration of about 0.5M with a pH of about 9.
  • said bivalent ion is magnesium chloride or magnesium sulphate at a concentration of about 3OmM.
  • said detergent is Nonidet-P40 at a concentration of about 0.5%.
  • said stabilizer is gelatin at a concentration of about 0.1%.
  • said sugar alcohol is sorbitol at a concentration of about 25mM.
  • said chloride containing monovalent ion is potassium chloride at a concentration about 0.1 M.
  • said buffer system is compatible with PCR inhibitory substances present in the sample.
  • the present invention also relates to a method for direct PCR amplification of samples, wherein the method comprises steps of:
  • said buffer system is used preferably to detect infections.
  • said samples are selected from a group comprising blood, plasma, serum and whole microbial cells.
  • said polymerase is Taq DNA, polymerase.
  • said buffer system is compatible with PCR inhibitory substances present in the sample.
  • said amplification is achieved for about 50% volume of blood sample, about 40% volume of plasma sample and even for about 70% volume of serum sample.
  • said method can be used to detect clinical pathogens present in the sample, preferably blood borne pathogens including their genetic sequences.
  • the method can be used to for direct PCR for whole microbial cells without nucleic acid isolation.
  • the buffer system used here may or may not contain chloride containing monovalent ion which is potassium chloride. While all the commercial buffers contain potassium chloride for PCR amplification.
  • Fig 1 Direct blood PCR for 16S ribosomal unit of Salmonella. Ll- 20% blood, L2- 30% blood, L3- 40% blood, L4- 50% blood.
  • Fig 2 Direct plasma PCR for 16S ribosomal unit of Salmonella. Ll -20% plasma, L2- 30% plasma, L3- 40% plasma, L4- 50% plasma, L5- Control reaction.
  • Fig 3 Direct plasma PCR for Hepatitis B virus core region. Ll- reaction with normal Taq buffer, L2 & L3- reaction with the unique buffer cocktail.
  • Fig 4 Direct serum PCR for 16 S ribosomal unit of Salmonella. Ll- 60% serum, L2- 70% serum. , •
  • Fig 5 Whole cell PCR for Salmonella and lambda phage DNA using unique buffer without potassium chloride.
  • blood sample refers to any kind of sample whose origin can be derived from blood. It can be, for example, liquid blood, such as fresh whole blood with all its constituents, or plasma. It also includes dried blood such as is present, for example, in bloodstains, to coagulated blood or the serum obtained there from.
  • the target nucleic acids to be amplified by the processes of the present invention can be present in the blood cells (e.g. genomic DNA, mRNA), in plasma, and in serum.
  • the nucleic acids can be the cell's own DNA or RNA which are liberated by cell lysis, or they can be foreign nucleic acids that are introduced by bacteria or viruses.
  • the present invention relates to the PCR amplification and detection of nucleic acids from blood, plasma, serum or any other samples with out nucleic acid extraction by employing a unique buffer formulation.
  • a major problem with diagnostic and forensic techniques based on PCR is the false- negative reactions or low sensitivity caused by inhibitory substances that interfere with PCR.
  • PCR analysis of blood samples which represents the largest fraction of human health related tests for diagnosis of genetic diseases, virus and microbial infections, blood typing, and safe blood banking.
  • Various studies indicate that the inhibitory effect of blood on PCR is primarily associated with direct inactivation of the thermostable DNA polymerase and/or capturing or degradation of the target DNA and primers. It has been reported that the protease activity in blood also contributes to the reduced efficiency of PCR.
  • the present invention gives a brief description of a unique buffer system for direct PCR without involving any nucleic acid isolation.
  • the buffer system helps in stabilizing the
  • PCR components from the inhibitory factors that are present in the blood serum. plasma or any other biological samples.
  • PCR can also be conducted for blood samples that are treated with anticoagulants such as heparin.
  • This buffer system is also useful for direct PCR from bacterial, viral or fungal cells without nucleic acid isolation.
  • One of the advantages of this system is that
  • PCR can be conducted effectively even in the presence of 50% volume of the blood sample, 40% volume of plasma sample and with 70% volume of the serum sample without any inhibition.
  • This present invention is useful in diagnostics particularly that involves detection of blood borne pathogens and direct PCRs from whole microbial cells.
  • the instant invention uses a universal buffer system which works for all kinds of blood samples. There is no need to adjust the concentration of the monovalent or bivalent or any kind of ions in the blood. Further, there is no requirement of employing few cycles of heating and cooling before carrying out the actual PCR reaction in the instant invention.
  • the present invention provides a method for the direct PCR amplification of nucleic acids from Blood, plasma, serum or whole microbial cells with out nucleic acid isolation by employing a unique buffer formulation.
  • This process is advantageous in that the blood sample need not be treated prior to amplification in order to isolate or to purify the target nucleic acid sequences.
  • the buffer system employed here is compatible with all the PCR inhibitory substances present in the blood. With this buffer formulation, amplification can be achieved even for 50% volume of the blood sample, 40% volume of plasma sample and with 70% volume of the serum sample. Whereas commercially available blood buffers report amplifications only for 20 to 30% blood.
  • the present invention is useful in diagnostic assays particularly those that detect viruses such as, e.g., cytomegalovirus, herpes simplex virus. Epstein-Barr virus, Hepatitis B or C virus and any blood-borne bacteria.
  • This invention may also be used to determine genetic sequences, for example, of humans, from blood and to identify foreign nucleic acids of microorganisms in the blood, including nucleic acids from bacteria, DNA or RNA viruses or eukaryotic nucleic acids.
  • the claimed process is particularly useful in the detection of small quantities of an infectious microorganism in any biological sample.
  • Example 1 Amplification of 16S ribosomal unit of Salmonella from infected whole blood and plasma
  • the 16S ribosomal unit of Salmonella bacterium was amplified from the blood, plasma or serum of clinical samples using the invented buffer system. Briefly, Salmonella infected blood, plasma or serum samples were subjected to PCR on Corbett palm cycler.
  • the PCR mixture (10 ⁇ l) included 1 ⁇ l of the unique buffer (10X consisting of Tris-HCl-0.5M; MgCl 2 or MgS0 4 -30 mM; gelatin-0.1%; Nonidet-P40-0.5%; sorbitol- 25 mM; KCl-0.1 M;), appropriate primers, dNTPS, Taq DNA polymerase and required amount of the template.
  • the conditions of the PCR were as follows: pre-denaturation at 94 C for 3 min and 30 cycles, with each cycle including denaturation at 94° C for 30 sec, annealing and extension at 55° C and 72° C for 30 sec and final extension at 72° C for 5 min. After the PCR. in order to extract the amplified product from the blood, plasma or serum pellet, water was added and extracted. The product expected was of 200 bp which was confirmed by agarose gel electrophoresis using a 1000 bp DNA ladder (Fig. 1,2 & 4).
  • Example 2 Amplification of antigenic core region of Hepatitis B virus from whole blood and plasma
  • the antigenic core region of Hepatitis B virus was amplified from plasma. Briefly, Hepatitis B infected plasma samples were subjected to PCR on Corbett palm cycler.
  • the PCR mixture (10 ⁇ l) included 1 ⁇ l of the unique buffer (10X consisting of Tris- HC1-0.5M; MgCl 2 or MgSo 4 -30 mM; gelatin-0.1%; Nonidet-P40-0.5%; sorbitol-25 mM; KCl-0.1 M;), appropriate primer, dNTPS, unique buffer, Taq DNA polymerase and required amount of the template
  • the conditions of the PCR were as follows: pre- denaturation at 94 0 C for 3 min and 35 cycles, with each cycle including denaturation at 94° C for 30 sec, annealing at 53° C for 45 sec, extension at 72° C for 1 min and final extension at 72° C for 7 min.
  • the expected product was of 550 bp length which was confirmed by agarose gel electrophor
  • Example 3 Amplification of Salmonella and lambda phage DNA using whole cells as template and using unique buffer without potassium chloride
  • PCR was conducted using whole cells of Salmonella and Lambda phage. Briefly, Salmonella and Lambda phage cells were subjected to PCR on Corbett palm cycler.
  • the conditions of the PCR were as follows: pre-denaturation at 94° C for 3 min and 30 cycles, with each cycle including denaturation at 94° C for 30 sec, annealing and extension at 55° C and 72° C for 30 sec and final extension at 72° C for 5 min.
  • the products expected were of 200 and 31 lbp for Salmonella and Lambda phage.
  • thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl. Environ. Microbiol. 64:3748-53.

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Abstract

The present invention relates to a buffer system for direct PCR amplification and a method thereof.

Description

A BUFFER SYSTEM AND A METHOD FOR DIRECT PCR AMPLIFICATION
FIELD OF THE INVENTION
This invention relates to the PCR amplification and detection of nucleic acids from blood, plasma, serum or any other whole microbial cells with out nucleic acid isolation by employing a unique buffer formulation. This system can be used for the detection of viruses such as Cytomegalovirus. Herpes simplex virus, Epstein-Ban- virus, Hepatitis B. or Hepatitis C virus and any blood-borne bacteria.
The present invention is related to direct PCR with blood, plasma, serum or any other whole microbial cells using a unique buffer formulation without the need for nucleic acid isolation. The buffer mentioned here is a universal buffer for any type of blood sample. This will find wide applications in diagnosis of clinical pathogens and also for direct PCRs from whole microbial cells.
BACKGROUND AND PRIOR ART OF THE INVENTION
Blood samples are extensively used for PCR-based diagnosis of microbial infections, genetic diseases, forensic analysis, as well as for blood banking. However, when applying nucleic acid amplification techniques to blood samples, the amplification capacity can be dramatically reduced or blocked by the presence of PCR-inhibitory substances. Inhibitors in blood, which have been identified, are either natural components of blood, mainly heme and leukocyte DNA, or added anticoagulants such as EDTA and heparin. Recently, immunoglobulin G present in human plasma was identified as a major inhibitor of diagnostic PCR in blood. Widely used thermostable polymerases like Thermus aquaticus DNA polymerase (Taq) and AmpliTaq Gold are completely inhibited in the presence of 0.004-0.2% whole human blood. Various agents have been tested for reducing the inhibitory effect of blood on Taq. It was found that an addition of betaine, bovine serum albumin, the single-stranded DNA binding protein of the T4 32 gene (gp 32), or a cocktail of protease inhibitors can partially relieve the blood inhibition and allow Taq to work in up to 2% blood (vol/vol), although this effect could be sample specific. Other complicating factors include EDTA and heparin, used as anti-coagulants, which can also inhibit DNA amplification. The addition of heparinase has been shown to counteract the heparin-mediated inhibition.
Conventional methods used in molecular biology to prepare nucleic acids from blood are complex and include steps such as centrifuging, phenol/chloroform extraction of the samples or precipitations of the nucleic acids with organic solvents, which are useless for rapid and possibly automatable enzymatic amplification of nucleic acids without substantial preparation. A recent compilation of such methods is found in "An efficient and simple method of DNA extraction from whole blood and cell-lines to identify infectious agents" by V. N. Loparev et al, J. Vir. Methods 34: 105-1 12 (1991) and in "Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material" by P.S. Walsh et al, BioTechniques 10: 506-513 (1991). Separation with a QIAamp kit followed by dialysis with a Millipore filter are required for eliminating the heme inhibition of hepatitis B virus detection. In addition, some of the above steps carry a risk of target DNA losses and are not suitable for automation. Moreover, even commercial kits specially formulated for DNA purification from blood samples such as QIAmp or GeneReleaser are not always satisfactory. The reason is due to an incomplete removal of Taq inhibitors, which can result in false-negative results. For example, 14% of the human blood samples tested for hepatitis B virus yielded false-negative results when using such blood kits.
United States Patent no 5501963 reports the use of different types of buffers depending on the concentration of the monovalent and bivalent ions present in the blood sample to achieve the PCR reaction. As an additional step to ensure efficient amplification the US patent 5501963 employs few cycles of heating and cooling before carrying out the actual PCR reaction.
Different methods of sample preparation have been developed to remove the inhibitory effect of blood. Despite the various advantages, these methods, in general are (a) time- consuming, (b) labor-intensive, (c) sample specific, and (d) run the potential risk of losing target microorganism or nucleic acids during processing. Further, they are not suitable for automation. Apart from the blood samples PCR can also be conducted with whole microbial cells without nucleic acid isolation using this buffer system. OBJECTS OF INVENTION
The main object of the present invention is to obtain a buffer system for direct PCR amplification.
Another main object of the present invention is to develop a method for direct PCR amplification of samples.
STATEMENT OF THE INVENTION
Accordingly, the present invention relates to a buffer system for direct PCR amplification, said buffer system comprises a buffer salt, a chloride or sulphate containing bivalent ion, a non-ionic detergent, a stabilizer and a sugar alcohol optionally alongwith a chloride containing monovalent ion and a method for direct PCR amplification of samples, wherein the method comprises steps of: (a) adding buffer system of claim 1, suitable primers, dNTP's and polymerase to the sample to obtain a PCR mixture; and (b) subjecting the PCR mixture to predetermined conditions for the amplification of nucleic acid in the sample.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a buffer system for direct PCR amplification, said buffer system comprises a buffer salt, a chloride or sulphate containing bivalent ion, a non-ionic detergent, a stabilizer and a sugar alcohol optionally alongwith a chloride containing monovalent ion.
In another embodiment of the present invention, said buffer system is used preferably to detect infections.
In yet another embodiment of the present invention, said samples are selected from a group comprising blood, plasma, serum and whole microbial cells. '
In still another embodiment of the present invention, said buffer is Tris-HCl at a concentration of about 0.5M with a pH of about 9. In still another embodiment of the present invention, said bivalent ion is magnesium chloride or magnesium sulphate at a concentration of about 3OmM.
In still another. embodiment of the present invention, said detergent is Nonidet-P40 at a concentration of about 0.5%.
In still another embodiment of the present invention, said stabilizer is gelatin at a concentration of about 0.1%.
In still another embodiment of the present invention, said sugar alcohol is sorbitol at a concentration of about 25mM.
In still another embodiment of the present invention, said chloride containing monovalent ion is potassium chloride at a concentration about 0.1 M.
In still another embodiment of the present invention, said buffer system is compatible with PCR inhibitory substances present in the sample.
The present invention also relates to a method for direct PCR amplification of samples, wherein the method comprises steps of:
a) adding buffer system of claim 1, suitable primers, dNTP's and polymerase to the sample to obtain a PCR mixture; and
b) subjecting the PCR mixture to predetermined conditions for the amplification of nucleic acid in the sample.
In another embodiment of the present invention, said buffer system is used preferably to detect infections.
In yet another embodiment of the present invention, said samples are selected from a group comprising blood, plasma, serum and whole microbial cells.
In still another embodiment of the present invention, said polymerase is Taq DNA, polymerase. In still another embodiment of the present invention, said buffer system is compatible with PCR inhibitory substances present in the sample.
In still another embodiment of the present invention, said amplification is achieved for about 50% volume of blood sample, about 40% volume of plasma sample and even for about 70% volume of serum sample.
In still another embodiment of the present invention, said method can be used to detect clinical pathogens present in the sample, preferably blood borne pathogens including their genetic sequences.
In still another embodiment of the present invention, the method can be used to for direct PCR for whole microbial cells without nucleic acid isolation. The buffer system used here may or may not contain chloride containing monovalent ion which is potassium chloride. While all the commercial buffers contain potassium chloride for PCR amplification.
BREIF DESCIPTION OF ACCOMPANYING DRAWINGS
Fig 1: Direct blood PCR for 16S ribosomal unit of Salmonella. Ll- 20% blood, L2- 30% blood, L3- 40% blood, L4- 50% blood.
Fig 2: Direct plasma PCR for 16S ribosomal unit of Salmonella. Ll -20% plasma, L2- 30% plasma, L3- 40% plasma, L4- 50% plasma, L5- Control reaction.
Fig 3: Direct plasma PCR for Hepatitis B virus core region. Ll- reaction with normal Taq buffer, L2 & L3- reaction with the unique buffer cocktail.
Fig 4: Direct serum PCR for 16 S ribosomal unit of Salmonella. Ll- 60% serum, L2- 70% serum. , •
Fig 5: Whole cell PCR for Salmonella and lambda phage DNA using unique buffer without potassium chloride.
The term "blood sample" mentioned here refers to any kind of sample whose origin can be derived from blood. It can be, for example, liquid blood, such as fresh whole blood with all its constituents, or plasma. It also includes dried blood such as is present, for example, in bloodstains, to coagulated blood or the serum obtained there from.
The target nucleic acids to be amplified by the processes of the present invention can be present in the blood cells (e.g. genomic DNA, mRNA), in plasma, and in serum. In plasma or serum, the nucleic acids can be the cell's own DNA or RNA which are liberated by cell lysis, or they can be foreign nucleic acids that are introduced by bacteria or viruses. The present invention relates to the PCR amplification and detection of nucleic acids from blood, plasma, serum or any other samples with out nucleic acid extraction by employing a unique buffer formulation.
A major problem with diagnostic and forensic techniques based on PCR is the false- negative reactions or low sensitivity caused by inhibitory substances that interfere with PCR. Of particular clinical importance is the PCR analysis of blood samples, which represents the largest fraction of human health related tests for diagnosis of genetic diseases, virus and microbial infections, blood typing, and safe blood banking. Various studies indicate that the inhibitory effect of blood on PCR is primarily associated with direct inactivation of the thermostable DNA polymerase and/or capturing or degradation of the target DNA and primers. It has been reported that the protease activity in blood also contributes to the reduced efficiency of PCR.
The present invention gives a brief description of a unique buffer system for direct PCR without involving any nucleic acid isolation. The buffer system helps in stabilizing the
PCR components from the inhibitory factors that are present in the blood; serum. plasma or any other biological samples. With this unique buffer system PCR can also be conducted for blood samples that are treated with anticoagulants such as heparin.
EDTA etc. This buffer system is also useful for direct PCR from bacterial, viral or fungal cells without nucleic acid isolation. One of the advantages of this system is that
PCR can be conducted effectively even in the presence of 50% volume of the blood sample, 40% volume of plasma sample and with 70% volume of the serum sample without any inhibition. This present invention is useful in diagnostics particularly that involves detection of blood borne pathogens and direct PCRs from whole microbial cells. The instant invention uses a universal buffer system which works for all kinds of blood samples. There is no need to adjust the concentration of the monovalent or bivalent or any kind of ions in the blood. Further, there is no requirement of employing few cycles of heating and cooling before carrying out the actual PCR reaction in the instant invention.
The present invention provides a method for the direct PCR amplification of nucleic acids from Blood, plasma, serum or whole microbial cells with out nucleic acid isolation by employing a unique buffer formulation. This process is advantageous in that the blood sample need not be treated prior to amplification in order to isolate or to purify the target nucleic acid sequences. The buffer system employed here is compatible with all the PCR inhibitory substances present in the blood. With this buffer formulation, amplification can be achieved even for 50% volume of the blood sample, 40% volume of plasma sample and with 70% volume of the serum sample. Whereas commercially available blood buffers report amplifications only for 20 to 30% blood. The present invention is useful in diagnostic assays particularly those that detect viruses such as, e.g., cytomegalovirus, herpes simplex virus. Epstein-Barr virus, Hepatitis B or C virus and any blood-borne bacteria. This invention may also be used to determine genetic sequences, for example, of humans, from blood and to identify foreign nucleic acids of microorganisms in the blood, including nucleic acids from bacteria, DNA or RNA viruses or eukaryotic nucleic acids. The claimed process is particularly useful in the detection of small quantities of an infectious microorganism in any biological sample.
The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of invention.
Example 1: Amplification of 16S ribosomal unit of Salmonella from infected whole blood and plasma
The 16S ribosomal unit of Salmonella bacterium was amplified from the blood, plasma or serum of clinical samples using the invented buffer system. Briefly, Salmonella infected blood, plasma or serum samples were subjected to PCR on Corbett palm cycler. The PCR mixture (10 μl) included 1 μl of the unique buffer (10X consisting of Tris-HCl-0.5M; MgCl2 or MgS04-30 mM; gelatin-0.1%; Nonidet-P40-0.5%; sorbitol- 25 mM; KCl-0.1 M;), appropriate primers, dNTPS, Taq DNA polymerase and required amount of the template. The conditions of the PCR were as follows: pre-denaturation at 94 C for 3 min and 30 cycles, with each cycle including denaturation at 94° C for 30 sec, annealing and extension at 55° C and 72° C for 30 sec and final extension at 72° C for 5 min. After the PCR. in order to extract the amplified product from the blood, plasma or serum pellet, water was added and extracted. The product expected was of 200 bp which was confirmed by agarose gel electrophoresis using a 1000 bp DNA ladder (Fig. 1,2 & 4).
Example 2: Amplification of antigenic core region of Hepatitis B virus from whole blood and plasma
The antigenic core region of Hepatitis B virus was amplified from plasma. Briefly, Hepatitis B infected plasma samples were subjected to PCR on Corbett palm cycler. The PCR mixture (10 μl) included 1 μl of the unique buffer (10X consisting of Tris- HC1-0.5M; MgCl2 or MgSo4-30 mM; gelatin-0.1%; Nonidet-P40-0.5%; sorbitol-25 mM; KCl-0.1 M;), appropriate primer, dNTPS, unique buffer, Taq DNA polymerase and required amount of the template The conditions of the PCR were as follows: pre- denaturation at 940C for 3 min and 35 cycles, with each cycle including denaturation at 94° C for 30 sec, annealing at 53° C for 45 sec, extension at 72° C for 1 min and final extension at 72° C for 7 min. The expected product was of 550 bp length which was confirmed by agarose gel electrophoresis using a 1000 bp DNA ladder (Fig. 3).
Example 3: Amplification of Salmonella and lambda phage DNA using whole cells as template and using unique buffer without potassium chloride
PCR was conducted using whole cells of Salmonella and Lambda phage. Briefly, Salmonella and Lambda phage cells were subjected to PCR on Corbett palm cycler. The PCR mixture (10 μl) included 1 μl of the unique buffer (10X consisting of Tris- HC1-0.5M; MgCl2 or MgSo4-30 mM; gelatin-0.1%; Nonidet-P40-0.5%; sorbitol-25 mM;), appropriate primers, dNTPS, Taq DNA polymerase and required amount of the template. The conditions of the PCR were as follows: pre-denaturation at 94° C for 3 min and 30 cycles, with each cycle including denaturation at 94° C for 30 sec, annealing and extension at 55° C and 72° C for 30 sec and final extension at 72° C for 5 min. The products expected were of 200 and 31 lbp for Salmonella and Lambda phage.
This was further confirmed by agarose gel electrophoresis using a 1000 bp DNA ladder (Fig. 5).
REFERENCES
1) Abu AI-Soud, W., L. J. Jδnsson, and P. Radstrom. 2000. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J. Clin. Microbiol. 38:345-350.
2) AI-Soud W A, Radstrom P. 2000. Effect of amplification facilitators on diagnostic PCR in the presence of blood, feces and meat. J. Clin. Microbiol. 38: 4463-70.
3) AI-Soud A W, Radstrom P. 1998. Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl. Environ. Microbiol. 64:3748-53.
4) Frackman S, Kobs G, Simpson D, Storts D. 1998. Betaine and DMSO: enhancing agents for PCR. Promega Notes 65:27.
5) Topal M D, Sinha N K. 1983. Products of bacteriophage T4 genes 32 and 45 improve the accuracy of DNA replication in vitro. J. Biol. Chem. 258:12274-79.
6) Kreader C A. 1996. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62: 1 102-06.
7) Rossen L, N.o slashed.skov P, Holmstr.o slashed.m K, Rasmussen O F.
1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA -extraction solution. Int. J. Food Microbiol. 17:37-45.
8) Izraeli S, Pfleiderer C, Lion T. 1991. Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood. Nucleic Acids Res.19:6051. 9) Loparev et al., "An efficient and simple method of DNA extraction from whole blood and cell-lines to identify infectious agents" by V. N. J. Vir. Methods 34: 105-1 12 (1991).
10) P. S. Walsh et al., "Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material" by BioTechniques 10(4):506-513
(1991).
11) Kramvis A, Bukovzer S, Kew M C 1996. Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, Genereleaser, and the phenol-chloroform method. J. Clin. Microbiol. 34:2731-33.
12) Lantz P-G, Al-Soud W A, Knutsson R, Hahn-Hgerdal B, Radstrom P.
2000. Biotechnical use of the polymerase chain reaction for microbial analysis of biological samples, p. 87-130. In M. R. El-Gewely (ed.), BIOTECHNOLOGY ANNUAL REVIEW, vol. 5. (Elsevier Science B.V., Amsterdam, The Netherlands).
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Claims

We Claim
1) A buffer system for direct PCR amplification, said buffer system comprises a buffer salt, a chloride or sulphate containing bivalent ion, a non-ionic detergent, a stabilizer and a sugar alcohol optionally alongwith a chloride containing monovalent ion.
2) The buffer system as claimed in claim 1, wherein said buffer system is used preferably to detect infections.
3) The buffer system as claimed in claim 1, wherein said samples are selected from a group comprising blood, plasma, serum and whole microbial cells.
4) The buffer system as claimed in claim 1, wherein said buffer is Tris-HCl at a concentration of about 0.5M with a pH of about 9.
5) The buffer system as claimed in claim 1, wherein said bivalent ion is magnesium chloride or magnesium sulphate at a concentration of about 3OmM.
6) The buffer system as claimed in claim 1, wherein said detergent is Nonidet- P40 at a concentration of about 0.5%.
7) The buffer system as claimed in claim 1, wherein said stabilizer is gelatin at a concentration of about 0.1%.
8) The buffer system as claimed in claim 1, wherein said sugar alcohol is sorbitol at a concentration of about 25mM.
9) The buffer system as claimed in claim 1, wherein said chloride containing monovalent ion is potassium chloride at a concentration about 0.1 M.
10) The buffer system as claimed in claim 1, wherein said buffer system is compatible with PCR inhibitory substances present in the sample.
11) A method for direct PCR amplification of samples, wherein the method comprises steps of: a) adding buffer system of claim 1, suitable primers, dNTP's and polymerase to the sample to obtain a PCR mixture; and
b) subjecting the PCR mixture to predetermined conditions for the amplification of nucleic acid in the sample.
12) The method as claimed in claim 1 1, wherein said buffer system is used preferably to detect infections.
13) The method as claimed in claim 1 1, wherein said samples are selected from a group comprising blood, plasma, serum and whole microbial cells.
14) The method as claimed in claim 11, wherein said polymerase is Taq DNA polymerase.
15) The method as claimed in claim 1 1, wherein said buffer system is compatible with PCR inhibitory substances present in the sample.
16) The method as claimed in claim 11, wherein said amplification is achieved for about 50% volume of blood sample, about 40% volume of plasma sample and even for about 70% volume of serum sample.
17) The method as claimed in claim 11, wherein said method can be used to detect clinical pathogens present in the sample, preferably blood borne pathogens including their genetic sequences.
PCT/IN2008/000477 2007-07-31 2008-07-30 A buffer system and a method for direct pcr amplification WO2009016652A1 (en)

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