Title of Invention

A BUFFER SYSTEM AND A METHOD FOR DIRECT PCR AMPLIFICATION

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

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, Epslein-Barr 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 fmd 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 ai, J. Vir. Methods 34:105-112 (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 QlAmp 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 30mM.
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 O.IM.
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 amplitication 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.
BREIF DESCIPTION OF ACCOMPANYING DRAWINGS
Fig 1: Direct blood PCR for 16S ribosomal unit oiSalmonella. LI- 20% blood, L2- 30% blood, L3- 40% blood, L4- 50% blood.
Fig 2: Direct plasma PCR for 16S ribosomal unit oi Salmonella. LI-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. LI- reaction with normal Taq buffer, L2 8c L3- reaction:with the unique buffer cocktail.
Fig 4: Direct serum PCR for. 16 S ribosomal unit of Salmonella. LI- 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 therrnostable 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, EDI A 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. The 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 paiticularly those that detect viruses such as, e.g., cytomegalovirus, herpes simplex virus, Epstew-Barr virus, HepaHiis B or C virus and an\' 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 \iY) included 1 \x\ of the unique buffer (lOX consisting of Tris-HCl-0.5M; MgCb or MgS04-30 mM; gelatin-0.1%; Nonidet-P40-0.5%; sorbitol-25 mM; KCI-OT 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: Ampliflcation 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 pi) included 1 pi of the unique buffer (lOX consisting of Tris-HCl-0.5M; MgCbor MgSo4-30 mM; gelatin-O.1%; Nonidet-P40-0.5%; sorbitol-25 mM; KCI-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'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 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 pi) included 1 pi of the unique buffer (lOX consisting of Tris-HCl-

0.5M; MgCl2 or MgSo4-30 rnM; 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 Ibp for Salmonella and Lambda phage. This was further confirmed by agarose gel electrophoresis using a 1000 bp DNA ladder (Fig. 5).
REFERENCES
1) Abu Al-Soud, W., L. J. Jonsson, 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) Al-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) Al-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. 62:1102-06.

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 t6 identify infectious agents" by V. N. J. Vir. Methods 34:105-112(1991).
JO) 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).
n)Kramvis A, Bukovzer S, KGW M C 1996. Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, Genereleaser, and the phenol-chloroform method. J. CHn. 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).
13) Altwegg M, Verhoef j 1995. Amplification methods in diagnostic microbiology. J Microbiol. Methods 23:3-138.
14) de Franchis R, Cross N C P, Foulkes N S, Cox T M. 1988. A potent inhibitor of Taq polymerase copurifies with human genomic DNA. Nucleic Acids Res. 16:10355.

15)Al-Soud W A, Radstrom P. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39:485-93.
16)Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K, 1994.
Identification of the heme compound copurified with deoxyribonucleic acid
(DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR)
'i amplification. J Forensic Sci. 39:362-72.
17)Morata P, Queipo-Ortuno I, Colmenero J. 1998. Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J. Clin. Microbiol. 36: 2443-46.
18) United States Patent No 5501963. 1996.


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 30mM.
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 O.IM.
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 11, wherein said buffer system is used preferably to detect infections.
13) The method as claimed in claim 11, wherein said samples are selected from a group comprising blood, plasma, serum and whole microbial cells.
14) The method as clairned in claim H, wherein said polymerase is Tag DNA polymerase.
15) The method as claimed in claim 11, 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 10% 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.

18) The buffer system and the method for direct PCR amplification as substantially herein described with reference to accompanying examples and figures.


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1672-che-2007 form-5 25-01-2011.pdf

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1672-che-2007 correspondence others.pdf

1672-che-2007 description (complete).pdf

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1672-che-2007 form-1.pdf

1672-che-2007 form-18.pdf

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Patent Number 249165
Indian Patent Application Number 1672/CHE/2007
PG Journal Number 41/2011
Publication Date 14-Oct-2011
Grant Date 07-Oct-2011
Date of Filing 31-Jul-2007
Name of Patentee BIGTEC PRIVATE LIMITED
Applicant Address J.K.TOWERS, SANGAM CIRCLE 46TH CROSS JAYANAGAR 8TH BLOCK BANGALORE 560 082
Inventors:
# Inventor's Name Inventor's Address
1 JAGANNATH MANJULA J.K.TOWERS, SANGAM CIRCLE 46TH CROSS JAYANAGAR 8TH BLOCK BANGALORE 560 082
2 PILLARISETTI VENKATA SUBBARAO J.K.TOWERS, SANGAM CIRCLE 46TH CROSS JAYANAGAR 8TH BLOCK BANGALORE 560 082
3 NAIR BHASKARAN CHANDRASEKHAR J.K.TOWERS, SANGAM CIRCLE 46TH CROSS JAYANAGAR 8TH BLOCK BANGALORE 560 082
PCT International Classification Number C12N15/10
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA