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EPICENTRE Forum 5 (3)
Detection of Blood-Borne Microbes with the MasterPure™ Complete DNA and RNA Purification Kit Judith T. Schanke, John Watson, and Haiying L. Grunenwald, Epicentre
Technologies Introduction The use of PCR for the detection of blood-borne microbes has increased significantly in recent years. Hepatitis C virus (HCV), present in human whole blood and serum samples, is now identified quickly and easily by isolating nucleic acids and subsequently amplifying a conserved region of the RNA virus by RT-PCR. For low copy number samples, a subsequent nested PCR amplification can generate sufficient product for accurate detection. Similarly, bacteria present in a human plasma sample are now detected by DNA isolation and subsequent amplification of a definitive bacterial gene. Here, we demonstrate the efficiency and ease of isolating nucleic acids from blood-derived samples using the new MasterPure™ Complete DNA and RNA Purification Kit.* Total nucleic acid, including both DNA and RNA, was isolated in less than one hour from HCV-containing whole blood and serum samples. The conserved 5' noncoding region of HCV, which is commonly used to classify HCV into genotypes and subtypes, was subsequently amplified using the MasterAmp™ RT-PCR Kit. Total nucleic acid was also efficiently isolated from human plasma that contained E. coli, as was demonstrated by the amplification of a region of the E. coli 16S rRNA gene. Methods and Results HCV serum samples Human serum samples containing HCV were obtained from Dr. Yury Khudyakov and Dr. Howard Fields, Hepatitis Branch, Centers for Disease Control, Atlanta, GA. Each sample was provided with a viral titer. These titers were used to calculate the maximum possible copy number of virus used in amplification reactions assuming 100% yield of viral RNA. Introduction of HCV into whole blood In order to simulate the isolation of HCV RNA from whole blood, two hundred microliters of non-infected whole blood were centrifuged and 75 µl of plasma were removed. The plasma was replaced with 75 µl of serum containing HCV RNA. This combined blood sample was then vortex mixed to resuspend the pelleted cells and the MasterPure Complete DNA and RNA Purification Kit was used to isolate nucleic acids from this whole blood sample. Rapid isolation of DNA and RNA from HCV-containing whole blood A total nucleic acid extraction was performed on a small aliquot (12.5 µl) of the HCV-containing whole blood sample using Epicentre's MasterPure Complete DNA and RNA Purification Kit according to the protocol described in Table 1. This volume of whole blood was used to facilitate nucleic acid isolation in a single 1.5-ml tube. This procedure is rapid and does not entail lysis and removal of the red blood cells before nucleic acid isolation. The precipitated nucleic acid obtained from this procedure, which contains both genomic DNA as well as total cellular and viral RNA, was resuspended in 25 µl of TE buffer. Five microliters of the sample were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized by transillumination (Figure 1A). The sample contains a prominent genomic DNA band. Ribosomal RNA bands are not apparent; however, the sample clearly contains viral RNA as demonstrated by the RT-PCR analysis described below.
Amplification of HCV RNA from whole blood nucleic acid isolation To verify that the HCV RNA had been isolated along with human total nucleic acid, the MasterAmp RT-PCR Kit was used to amplify a 296 bp region of the highly conserved 5' noncoding region of HCV. The RT-PCR reaction contained 5 µl (containing 300-400 copies of HCV) of the isolated total nucleic acid sample, 1X RT-PCR Buffer, 3 mM MgCl2, 0.5 mM MnSO4, 2X MasterAmp PCR Enhancer (with betaine+), 12.5 pmoles each of HCVRT-F and HCVRT-R primers (Table 2), 400 µM each dNTP, and 2.5 units of RetroAmp™ RT DNA Polymerase. Reverse transcription was performed at 60°C for 20 minutes, followed by 40 cycles of 92°C for 30 seconds, 55°C for 1 minute, and 72°C for 1 minute. Ten microliters of the RT-PCR product were separated on a 2% agarose gel, stained with SYBR® Gold (Molecular Probes), and visualized by transillumination (Figure 1B). The 296 bp product amplified from approximately 300 copies of HCV was easily detectable.
Abundant product yield obtained from nested amplification of the HCV 5' noncoding region For further amplification and to increase confidence in the detection of the HCV product, a second, nested PCR was performed using primers internal to the HCVRT primers used above for the RT-PCR. The RT-PCR product was diluted 1:10 or 1:100 in water and 1 µl of each dilution was used in a nested PCR. Each reaction contained 1X PCR buffer, 2.5 mM MgCl2, 2X MasterAmp PCR Enhancer (with betaine), 200 µM each dNTP, 12.5 pmoles each of HCVIN-F and HCVIN-R primers (Table 2), 1 µl of each diluted RT-PCR product, and 1.25 units of MasterAmp Taq DNA Polymerase in a total volume of 25 µl. These nested reactions were amplified with 20 cycles of the following profile: 94°C for 30 seconds, 64°C for 1 minute, and 73°C for 1 minute. Two microliters (8%) of each product were separated by agarose gel electrophoresis. The gel was stained with SYBR Gold and visualized by transillumination. The prominent HCV nested amplification products were obtained even with a 100-fold template dilution (Figure 1C). In some cases of low viral titers, it may be necessary to perform such a nested PCR to detect the presence of the virus. Abundant amplification of HCV RNA purified from clinical serum samples Typically, viruses such as HCV are isolated from human serum instead of whole blood, as was performed above. The MasterPure Complete DNA and RNA Purification Kit was used to isolate total nucleic acid from 50 µl of each of two HCV-containing serum samples. The resulting nucleic acid pellet, containing cellular DNA and total cellular and viral RNA, was resuspended in 50 µl of TE buffer. Five microliters of each sample (containing approximately 800-900 copies of HCV) were then subjected to RT-PCR using the conditions described above, but using only 30 cycles for the amplification. Ten percent of each reaction was separated by agarose gel electrophoresis, stained with SYBR Gold, and visualized by transillumination. The 296-bp amplification product of the 5' noncoding region of HCV is readily detectable when isolated from the serum (Figure 2).
Detection of bacterial DNA in human plasma Cells from a single E. coli colony were used to introduce intact E. coli into 50 µl of human plasma. This model system was created to determine if the MasterPure Complete Kit could be used to isolate both the human and E. coli DNA, and to demonstrate that the E. coli DNA could be detected in the nucleic acid mixture. Total nucleic acid was isolated from the plasma sample using the standard protocol of the MasterPure Complete DNA and RNA Purification Kit.1 The pellet, containing both DNA and RNA, was resuspended in 50 µl of TE buffer. Fifteen microliters of the sample were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized by transillumination. The pure, high molecular weight genomic DNA and the prominent ribosomal RNA isolated can be seen in Figure 3A. One microliter (2% of the DNA isolated) of the human plasma total nucleic acid was used to amplify a 350 bp region of the 16S ribosomal gene of E. coli. One nanogram of purified E. coli DNA was used as a positive control for PCR amplification. The amplification reactions included 1X PCR buffer, 3 mM MgCl2, 1X MasterAmp PCR Enhancer (with betaine), 200 µM each dNTP, 50 pmoles of each 16S rRNA primer (Table 2), 1.25 units of MasterAmp Taq DNA Polymerase, and 1 µl of isolated sample or 1 ng of E. coli DNA control template. A reaction with no DNA template, a negative control for the detection of E. coli DNA contamination, was also performed. The samples were heated at 94°C prior to addition of MasterAmp Taq Polymerase. Then the reactions were denatured for 4 minutes at 94°C and cycled 25 times with the following conditions: 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. Five microliters of each sample were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized by transillumination (Figure 3B). The 350 bp product is clearly detectable in both the positive control and the E. coli-contaminated human DNA sample, but not in the no DNA control. The MasterPure Complete DNA and RNA Purification Kit was used to successfully isolate E. coli genomic DNA from human plasma.
Summary Nucleic acid from HCV-containing whole blood samples can be purified with the MasterPure Complete DNA and RNA Purification Kit in less than one hour, and HCV RNA can be easily detected from the resulting total nucleic acid sample with Epicentre's MasterAmp RT-PCR Kit. HCV RNA can be detected similarly from human serum samples. Performing a second nested PCR on the HCV RT-PCR product using MasterAmp Taq DNA Polymerase improves the sensitivity of virus detection. In addition, bacterial DNA can easily be amplified from nucleic acid isolated from E. coli-contaminated plasma samples isolated with the MasterPure Complete DNA and RNA Purification Kit; therefore blood-borne microbes would likely be detectable from these samples using PCR.
More Information MasterPure™ Complete DNA and RNA Purification Kit MasterAmp™ High Fidelity RT-PCR Kit |
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