Method for purifying nucleic acids

Details Method for purifying nucleic acids Method for purifying nucleic acids

1999-03-16

2001-08-14

Campbell, Eggerton A. (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120

Reexamination Certificate

active

06274308

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to methods for purifying nucleic acids from biological samples containing nucleic acids and other materials. More specifically, the invention relates to methods for rapid and automatable purification of viral RNA from cell free biological samples.
Contamination of the blood supply with pathogenic viruses such as human immunodeficiency virus (HIV) and hepatitis has become an increasingly serious problem. The prevailing opinion in the NIH and elsewhere is that within 2-5 years all blood should be screened using polymerase chain reaction (PCR) analysis in addition to serological tests. It is thought that this will prevent at least one hundred transfusion-associated cases of hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV per year. Serological tests were until recently the method of choice for screening blood. Serological tests detect the presence of antibodies raised against viral agents in the blood. These tests, while easy to perform and efficient, have the drawback of not being able to detect an infection if an antibody response is not mounted. It is, therefore, difficult to use the tests to detect individuals in the early stages of infection. Because of this and other practical limitations to serological testing, there is a real need for methods that will detect infection during the window period before the standard serological tests are viable. Isolating viral nucleic acids present in the blood plasma followed by PCR amplification enables the detection of virus at the earliest stages of infection. The detection of virus at these stages is crucial to insure that the blood supply is free from contamination.
The screening of blood and related biological materials in the medical setting is performed on a massive scale. If we include the screening of plasma for the preparation of blood derivatives, around 20 million tests are done per year. Blood centers commonly test as much as one thousand units of blood each day. The preparation of isolated nucleic acids from a thousand samples of blood per day using the presently available techniques would require a huge staff of technicians. There is clearly a great need for rapid automatable methods for the detection of viral RNA during the early stages of infection to safeguard the blood supply against viral contamination.
Purifying viral RNA has been complicated in the past by the need for time-consuming procedures such as organic extractions and precipitation steps. There has long been a need for simple and efficient methods to purify RNA. The standard procedures for the purification of either RNA or DNA involve the solubilization of cells or virions either by the use of chaotropic ions such as guanidinium isothiocyanate (GuSCN) or by the dissolution of proteins by proteinase K. Solubilization is then followed by nucleic acid purification steps such as phenol/chloroform extraction, alcohol precipitation, and washing (Chomczynski et al. 1987). Methods lacking the above-mentioned nucleic acid purification steps have been found to suffer from interference by inhibitors of the enzymes used in PCR that are present in many sera or, in our experience, give false negative results when tested on undiluted samples (Ali et al. 1993; Ravaggi et al. 1992; Lai et al. 1994; Hayashi et al. 1994). Thus, the majority of existing methods are unsuitable for automated PCR because of either the necessity of performing multiple steps or their general unreliability.
One currently available method and kit for the purification of certain viral RNA from plasma uses a silica gel-based membrane (QIAGEN News 1995). However, this method is specifically adapted to purification of the RNA from a single virus, i.e., HCV, and further involves numerous centrifugations and is therefore not amenable to automation. An automated RNA purification system based on the use of silica-gel based membranes is also available (QIAGEN Catalogue 1997).
In view of the above considerations, it is clear that simpler and faster methods of RNA purification are needed. Methods compatible with automation are especially sought after.
Accordingly, it is one of the purposes of this invention to overcome the above limitations in the purification of RNA of viral origin by providing a method that enables the automation of the step of extraction of nucleic acids from serum or plasma. An RNA purification method suitable for automation should have a solubilization step, which dissolves cells and viruses and quantitatively liberates the RNA while inactivating, or at least inhibiting, ribonuclease (RNase). In addition, since centrifugation is difficult to incorporate into automated technology, the method should include a capture step in which the desired nucleic acid can be specifically or non-specifically bound to a solid phase permitting inhibitors to be removed by washing.
SUMMARY OF THE INVENTION
It has now been discovered that these and other objectives can be achieved by the present invention, which provides a method for purifying nucleic acids of viral origin. The method allows for the rapid purification of viral RNA from biological samples and is compatible with automation and nucleic acid amplification techniques such as RT-PCR (reverse transcription-PCR). Although a preferred biological sample is blood plasma or serum, the method is compatible with a variety of biological samples from mammalian, bacterial, yeast and plant sources.
In one embodiment the invention is a method for purifying viral RNA, comprising:
(a) applying a biological sample containing viral RNA to a hydrophilic polyvinylidine fluoride (PVDF) membrane which contains pores having an average diameter less than about 0.45 &mgr;m;
(b) passing the biological sample through the PVDF membrane;
(c) washing the membrane to remove impurities while selectively retaining the viral RNA;
(d) suspending the viral RNA using an eluant to provide purified viral RNA; and
(e) recovering the purified viral RNA.
Preferably, the membrane contains pores having an average diameter of from about 0.1 &mgr;m to about 0.3 &mgr;m, more preferably from about 0.15 &mgr;m to about 0.25 &mgr;m, and still more preferably, about 0.22 &mgr;m. The membrane is also preferably low protein-binding.
The passing step can comprise applying a pressure differential across the membrane, such as applying negative pressure below the membrane, or applying positive pressure above the membrane. Alternatively, the passing step can comprise applying centrifugal force to promote flow of the biological sample across the membrane.
The method can further comprise lysing the biological sample before the applying step.
The method is capable of purifying viral RNA having a length less than about 40,000 nucleotides, and is effective to purify viral RNA having a length less than about 30,000 nucleotides. The viral RNA is preferably single stranded. The method is well suited to purify viral RNA from a virus selected from the group consisting of hepatitis C virus, hepatitis A virus, hepatitis G virus, human immunodeficiency virus, human T-cell leukemia virus I, human T-cell leukemia virus II, and human lymphotropic virus.
The method can further comprise removing cellular components of the biological sample prior to applying the sample to the membrane. For example, if the biological sample is a blood sample, the removing step can comprise removing blood cells from the sample.
Alternatively, if the viral RNA is to be obtained from a cell culture, the removing step can comprise removing the cultured cells from the culture medium, to leave a supernatant substantially free of cells. Removal of cells can be accomplished by centrifugation or by a separate filtration step.
In the method, it is preferred that the biological sample be substantially free of cellular components. Especially preferred biological samples include serum or plasma.
The biological sample can contain whole virus. For example, the whole virus can be selected from the group consisting of hepatitis C virus, hepatitis A virus, hepatitis G virus, human immunodeficie

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