USE OF RNASE H FOR THE SELECTIVE AMPLIFICATION OF VIRAL DNA

Abstract

The present invention concerns the amplification of at least a first and a second target nucleic acid that may be present in at least one fluid sample using RNase H and a polymerase with reverse transcriptase activity.

Claims

1. A process for amplifying a DNA target nucleic acid that may be present in a sample, the process comprising the steps of contacting the DNA target nucleic acid from the sample with an amplification reagent comprising RNase H and a polymerase with reverse transcriptase activity; incubating in a reaction vessel the DNA target nucleic acid with the amplification reagent for a period of time, under conditions suitable for transcription of RNA by the polymerase with reverse transcriptase activity to occur; and incubating in the reaction vessel the DNA target nucleic acids with the amplification reagent for a period of time, under conditions sufficient for an amplification reaction indicative of the presence or absence of the DNA target nucleic acid to occur.

2. The process of claim 1 wherein said polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to a respective wildtype polymerase.

3. The process of claim 2 wherein said polymerase with reverse transcriptase activity comprising a mutation is selected from the group consisting of a CS5 DNA polymerase, a CS6 DNA polymerase, a Thermotoga maritima DNA polymerase, a Thermus aquaticus DNA polymerase, a Thermus thermophilus DNA polymerase, a Thermus flavus DNA polymerase, a Thermus filiformis DNA polymerase, a Thermus sp. sps17 DNA polymerase, a Thermus sp. Z05 DNA polymerase, a Thermotoga neapolitana DNA polymerase, a Termosipho africanus DNA polymerase, and a Thermus caldophilus DNA polymerase.

4. The process of claim 3 wherein said polymerase with reverse transcriptase activity comprising a mutation is from Thermus sp. Z05 DNA polymerase.

5. The process of claim 1, further comprising amplifying a RNA target nucleic acid that may be present in said sample.

6. The process of claim 1 wherein said DNA target nucleic acid is Hepatitis B Virus (HBV).

7. An automated process for amplifying and determining the quantity of a DNA target nucleic acid that may be present in a fluid sample, said process comprising the steps of incubating in a reaction vessel said DNA target nucleic acid with a solution comprising amplification reagents, and oligonucleotides specific for said DNA target nucleic acid, wherein said amplification reagents comprise RNase H and a polymerase with reverse transcriptase activity, for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur; incubating in the reaction vessel the DNA target nucleic acid with the solution comprising amplification reagents, and oligonucleotides specific for the DNA target nucleic acid for a period of time and under conditions sufficient for an amplification reaction indicative of the presence or absence of the DNA target nucleic acid to occur; and determining the quantity of the DNA target nucleic acid by referencing to an external calibration or by implementing an internal quantitative standard.

8. The automated process of claim 7 wherein said polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to a respective wildtype polymerase.

9. The automated process of claim 8 wherein said polymerase with reverse transcriptase activity comprising a mutation is selected from the group consisting of a CS5 DNA polymerase, a CS6 DNA polymerase, a Thermotoga maritima DNA polymerase, a Thermus aquaticus DNA polymerase, a Thermus thermophilus DNA polymerase, a Thermus flavus DNA polymerase, a Thermus filiformis DNA polymerase, a Thermus sp. sps17 DNA polymerase, a Thermus sp. Z05 DNA polymerase, a Thermotoga neapolitana DNA polymerase, a Termosipho africanus DNA polymerase, and a Thermus caldophilus DNA polymerase.

10. The automated process of claim 9 wherein said polymerase with reverse transcriptase activity comprising a mutation is from Thermus sp. Z05 DNA polymerase.

11. The automated process of claim 7, further comprising amplifying a RNA target nucleic acid that may be present in said sample.

12. The automated process of claim 7 wherein said DNA target nucleic acid is Hepatitis B Virus (HBV).

Description

DESCRIPTION OF THE FIGURES

[0248] FIG. 1:

[0249] Schematic depiction of the sample preparation workflow as used in an embodiment of the invention. Arrows pointing down denote addition of a component or reagent to each respective well of the deepwell plate mentioned above, arrows pointing up their respective removal. These actions were performed manually in steps 2, 3, 4, 21 and 22, by the process head of the apparatus in steps 10, 14, 16, 18, and 24, and by the reagent head of the apparatus in steps 5, 6, 7, 11, 15 and 19.

[0250] It has to be understood that the volumes used can be adjusted flexibly within the spirit of the invention, for example at least about up to 30% of the disclosed values. In particular, in the case of step 2, the sample volume can be variable in order to take into account the different types of fluid samples which may require more or less starting material for obtaining proper results, as known by the artisan. For example, the range is from about 100 ul to about 850 ul. Or, it is about 100 ul, about 500 ul or about 850 ul. For example, the volume in the respective vessels is adjusted to an identical total volume with the diluent in step 3. For example, as in the scheme shown in FIG. 1, the total volume adds up to about 850 ul.

[0251] FIG. 2A-G:

[0252] Growth curves of the amplifications of the target nucleic acids derived from HIV, HBV and CT carried out on a LightCycler480 (Roche Diagnostics GmbH, Mannheim, Del.) as described in

[0253] Example 1. The “Signal” indicated on the y-axis is a normalized fluorescent signal. The x-axis shows the number of the respective PCR cycle.

[0254] The growth curves of HIV and HBV are shown along with the growth curves of the corresponding internal control nucleic acid. The respective target nucleic acid curves are represented by straight lines, the control nucleic acid curves by dotted lines.

[0255] FIG. 2A: Qualitative HIV assay, measured in the channel for detection of the target probe.

[0256] FIG. 2B: Qualitative HIV assay, measured in the channel for detection of the control probe.

[0257] FIG. 2C: Quantitative HIV assay, measured in the channel for detection of the target probe.

[0258] FIG. 2D: Quantitative HIV assay, measured in the channel for detection of the control probe.

[0259] FIG. 2E: Quantitative HBV assay, measured in the channel for detection of the target probe.

[0260] FIG. 2F: Quantitative HBV assay, measured in the channel for detection of the control probe.

[0261] FIG. 2G: CT assay, measured in the channel for detection of the target probe.

[0262] FIG. 3:

[0263] Linearity of the quantitative HBV assay in EDTA plasma according to the data in Example 2.

[0264] FIG. 4:

[0265] Linearity of the quantitative HBV assay in serum according to the data in Example 2.

[0266] FIG. 5:

[0267] Linearity of the quantitative HCV assay in EDTA plasma according to the data in Example 2.

[0268] FIG. 6:

[0269] Linearity of the quantitative HCV assay in serum according to the data in Example 2.

[0270] FIG. 7:

[0271] Linearity of the quantitative HIV assay in EDTA plasma according to the data in Example 2.

EXAMPLES

[0272] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

[0273] This example describes a process for isolating and simultaneously amplifying at least a first and a second target nucleic acid using a single generic internal control nucleic acid.

[0274] In brief, in the depicted embodiment, realtime PCR is carried out simultaneously and under identical conditions on a panel of several different targets comprising bacteria (Chlamydia trachomatis, CT) as well as a DNA virus (HBV) and an RNA virus (HIV). All samples were processed and analyzed within the same experiment, i.e. on the same deepwell plate (for sample preparation) or multiwell plate (for amplification and detection), respectively.

[0275] The following samples were prepared and subsequently analyzed:

TABLE-US-00001 Reagent Manufacturer: HIV-1M Secondary Standard, 50′000 cp/ML Roche HBV Secondary Standard, 400 IU/ml Roche CT (DNA POS CTL pCHL-1) Roche

[0276] Suitable standards or other types of targets are available to the skilled artisan.

[0277] The instruments listed in the following table were used according to the instructions of the respective manufacturer:

TABLE-US-00002 Instrument Manufacturer Hamilton Star Hamilton Medical AG (Bonaduz, CH) Light Cycler 480 Roche Diagnostics GmbH (Mannheim, DE) Chameleon Sealer K biosystems (Essex, UK) Compressor K biosystems (Essex, UK)

[0278] For sample preparation the following reagents were used as diluents:

TABLE-US-00003 Reagent Manufacturer: PreservCyt Thin Prep K3 EDTA Plasma, PCR neg. Roche

[0279] The following dilutions were prepared in advance and stored overnight (plasma dilutions at −60 to −90° C., PreservCyt dilutions at 2-8° C.):

TABLE-US-00004 Target Concentration Matrix HBV 50 IU/ml K3 EDTA plasma HIV-1M 100 cp/ml K3 EDTA plasma CT 2.5 fg/ml PreservCyt

[0280] Each respective sample (500 ul) and each respective specimen diluent (350 ul) were pipetted manually into a deepwell plate, wherein each sample was added to three different wells for triplicate analysis. To each well containing an HIV or HBV sample, 50 ul of an internal control nucleic acid were manually added. For the qualitative HIV assay, an RNA serving as a qualitative control was added (100 armored particles/sample). For the quantitative HIV assay, an RNA serving as a quantitative standard was added (500 armored particles/sample). For the quantitative HBV assay, a DNA serving as a quantitative standard was added (1E4 copies/sample). The sequence of said control nucleic acids was identical in all cases and selected from the group of SEQ ID NOs 45-48.

[0281] The respective control nucleic acid was stored in the following buffer:

TABLE-US-00005 IC/IQS - Storage Buffer Conc. or pH Tris (mM) 10 EDTA (mM) 0.1 Sodium Azide (w/v, %) 0.05 Poly rA RNA (mg/l) 20 pH 8

[0282] Sample preparation was performed on a Hamilton Star (Hamilton, Bonaduz, CH), following the workflow according to the scheme depicted in FIG. 1 and using the following reagents:

TABLE-US-00006 Protease reagent Conc. or pH Tris (mM) 10 EDTA (mM) 1 Calcium Chloride (mM) 5 Calcium Acetate (mM) 5 Esperase (mg/ml) 80 Glycerin (w/v, %) 50 pH 5.5

TABLE-US-00007 MGP Reagent Conc. or pH MPG Powder (mg/ml) 60 Tris (mM) 30 Methylparaben (w/v, %) 0.1 Sodium Azide (w/v, %) 0.095 pH 8.5

TABLE-US-00008 Lysis Reagent Conc. or pH Guanidine Thiocyanate (M) 4 Sodium Citrate (mM) 50 Polydocanol (w/v, %) 5 Dithiotreitol (w/v, %) 2 pH 5.8

TABLE-US-00009 Wash buffer Conc. or pH Sodium Citrate (mM) 7.5 Methylparaben (w/v, %) 0.1 pH 4.1

TABLE-US-00010 Elution buffer Conc. or pH Tris (mM) 30 Methylparaben (w/v, %) 0.2 pH 8.5

[0283] After the final step, the process head of the Hamilton Star apparatus added the respective mastermixes (Mmxs) containing amplification reagents to each well, mixed the fluids containing the isolated nucleic acids with the Mmx and transferred each resulting mixture to a corresponding well of a microwell plate in which the amplification was carried out.

[0284] The following mastermixes (each consisting of the two reagents R1 and R2) were used:

[0285] For HIV:

TABLE-US-00011 Concentration/ 50 μl-PCR [μM] R1 Reagent Water (PCR grade) Mn(Ac).sub.2 * 4H.sub.2O (pH 6.1 adjusted with Acetic Acid) 3′000 NaN3/Ri, buffered with 10 mM Tris at pH 7 [%] 0.018 R2 Reagent DMSO [%] 5.000% NaN3/Ri, buffered with 10 mM Tris at pH 7 [%] 0.027% Potassium acetate pH 7.0 110′000  Glycerol [%] 3.000% Tricine pH 8.0 50′000  Igepal [%] 0.024% dGTP 337.5 dATP 337.5 dCTP 337.5 dUTP 675 Primers/probes selected from SEQ ID NOs 1-35 0.1-0.15 SEQ ID NO 42 0.1 SEQ ID NO 43 0.1 SEQ ID NO 44 0.1 Uracil-N-Glycosylase 10 (U/reaction)  Z05-D Polymerase 40 (U/reactionn) NTQ21-46A-Aptamer 0.222 Water

[0286] For HBV:

TABLE-US-00012 Concentration/ 50 μl-PCR R2 Reagent H20 100 % Tricine 7.7 40 mM Tween 0.03 % (v/v) Glycerol 5 % (v/v) KOH 25.2 mM KOAc 121.8 mM NTQ21-46A (Aptamer) 0.2625 uM dGTP 0.42 uM dATP 0.42 uM dCTP 0.42 uM dUTP 0.84 uM SEQ ID NO 36 1.2 uM SEQ ID NO 37 0.1 uM SEQ ID NO 38 1.2 uM SEQ ID NO 42 0.6 uM SEQ ID NO 43 0.6 uM SEQ ID NO 44 0.15 uM Z05D Polymerase 35 (U/reaction) Uracil-N-Glycosylase 2 (U/reaction) Sodium Azide 0.027 % (m/v) R1 Reagent H20 100 % MgOAc 2.5 mM MnOAc pH 6.1 2.5 mM Sodium Azide 0.018 % (m/v)

[0287] For CT:

TABLE-US-00013 Concentration/ 50 μl-PCR R1 Reagent Water (PCR grade) Mn(Ac).sub.2 (pH 6.5 in 0.002% (V/V) Glacial Acetic 2.7 mM Acid) NaN3 0.0135% (W/V) R2 Reagent NaN3/Ri, buffered with 10 mM Tris at pH 7 [%] 0.0315% Potassium acetate 112.4 mM Glycerol [%]   3.5% Tricine 61 mM Potassium hydroxide 28.4 mM dGTP 525 uM dATP 525 uM dCTP 525 uM dUTP 1.05 mM SEQ ID NO 39 750 nM SEQ ID NO 40 600 nM SEQ ID NO 41 116 nM Aptamer NTQ-46A 175 nM Uracil-N-Glycosylase 5 U/reaction Z05-D Polymerase 31 U/reaction

[0288] For amplification and detection, the microwell plate was sealed with an automated plate sealer (see above), and the plate was transferred to a LightCycler 480 (see above).

[0289] The following PCR profile was used:

TABLE-US-00014 Thermo cycling profile Acqui- Ramp Anal- Program Target sition Hold Rate ysis Name (° C.) Mode (hh:mm:ss) (° C./s) Cycles Mode Pre-PCR 50 None 00:02:00 4.4 1 None 94 None 00:00:05 4.4 55 None 00:02:00 2.2 60 None 00:06:00 4.4 65 None 00:04:00 4.4 1st 95 None 00:00:05 4.4 5 Quanti- Measure- 55 Single 00:00:30 2.2 fication ment 2nd 91 None 00:00:05 4.4 45 Quanti- Measure- 58 Single 00:00:25 2.2 fication ment Cooling 40 None 00:02:00 2.2 1 None

TABLE-US-00015 Detection Format (Manual) Filter Combination Integration Time (sec) 435-470 1 495-525 0.5 540-580 0.5 610-645 0.5 680-700 1

[0290] The Pre-PCR program comprises initial denaturing and incubation at 55, 60 and 65° C. for reverse transcription of RNA templates. Incubating at three temperatures combines the advantageous effects that at lower temperatures slightly mismatched target sequences (such as genetic variants of an organism) are also transcribed, while at higher temperatures the formation of RNA secondary structures is suppressed, thus leading to a more efficient transcription.

[0291] PCR cycling is divided into two measurements, wherein both measurements apply a one-step setup (combining annealing and extension). The first 5 cycles at 55° C. allow for an increased inclusivity by pre-amplifying slightly mismatched target sequences, whereas the 45 cycles of the second measurement provide for an increased specificity by using an annealing/extension temperature of 58° C.

[0292] Using this profile on all samples comprised on the microwell plate mentioned above, amplification and detection was achieved in all samples, as depicted in FIG. 2. This shows that the sample preparation prior to amplification was also successfully carried out.

[0293] The results for the qualitative and quantitative HIV internal controls and the quantitative HBV internal control are depicted separately in FIG. 2 for the sake of clarity. It can be seen that the controls were also successfully amplified in all cases. The quantitation of the HIV and HBV targets in the quantitative setup were calculated by comparison with the internal control nucleic acid serving as a quantitative standard.

Example 2

[0294] The generic amplification process described hereinabove was carried out on a variety of different target nucleic acids in separate experiments but under identical conditions. Isolation of the respective nucleic acid was carried out as described under Example 1.

[0295] The respective generic internal control nucleic acid was selected from SEQ ID NOs 45-49 and was armored RNA for RNA targets and lambda-packaged DNA for DNA targets. For qualitative RNA assays, 300 particles were added per sample, for quantitative RNA assays 3000 and for all DNA assays 500.

[0296] The following PCR profile was used on all targets:

TABLE-US-00016 Tar- Acqui- Ramp get sition Plateau Measure Rate [° C.] Mode [hh:mm:ss] [hh:mm:ss] [° C./s] Pre- UNG- 50 none 00:02:00 00:00:00 2.2 PCR Step UNG/ 94 none 00:00:05 00:00:00 4.4 Template Denatur- ation RT-Step 55 none 00:02:00 00:00:00 2.2 60 none 00:06:00 00:00:00 4.4 65 none 00:04:00 00:00:00 4.4 1st 95 none 00:00:05 00:00:00 4.4 Measurement 55 single 00:00:30 00:00:08 2.2 2nd 91 none 00:00:05 00:00:00 4.4 Measurement 58 single 00:00:25 00:00:08 2.2 Cooling 40 none 00:02:00 00:00:00 2.2

TABLE-US-00017 Name Cycles Pre-PCR 1 1st Measurement 5 2nd Measurement 45 Cooling 1

[0297] In detail, the following experiments were performed:

[0298] 1. Qualitative multiplex analysis of HBV, HCV and HIV

[0299] a. Mastermix

TABLE-US-00018 R1: Conc. in 50 ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH: 6.41 R2: Conc. in Reagent 50 ul-PCR (uM) DMSO (%) 5.4 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.027 KOAc (pH 7.0) 120′000 Glycerol (%) 3 Tween 20 (%) 0.015 Tricine pH 8.0  60′000 NTQ21-46A - Aptamer 0.2222 Uracil-N-Glycosylase (U/uL) 0.2 dGTP 400.0 dATP 400.0 dCTP 400.0 dUTP 800.0 ZO5-D Polymerase (U/ul)* 0.9 Primers/probes selected from SEQ ID NOs 1-35 0.125-0.3  SEQ ID NO 36 0.100 SEQ ID NO 37 0.100 SEQ ID NO 38 0.150 Primers/probes selected from SEQ ID NOs 60-76 0.050-0.250 SEQ ID NO 42 0.200 SEQ ID NO 43 0.200 SEQ ID NO 44 0.100

[0300] Analytical Sensitivity/LOD

[0301] For each detected virus (HIV-1 group M, HIV-1 group 0, HIV-2, HBV and HCV), at several concentrations/levels at and around the anticipated LOD for EDTA-plasma. One panel per virus and concentration was tested with at least 20 valid replicates per concentration. The LOD was determined by PROBIT analysis (see Table 1-5).

[0302] HIV

TABLE-US-00019 TABLE 1 HIV-1 Group M Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 32 cp/mL  21 21 100%  16 cp/mL  21 21 100%  8 cp/mL 21 21 100%  4 cp/mL 21 20 95% 2 cp/mL 21 15 71% 1 cp/mL 21 9 43% 0 cp/mL (neg. control) 12 0  0% LOD by PROBIT analysis (95% hitrate) 4.06 cp/mL 95% confidence interval for LOD by PROBIT 2.85-9.24 analysis cp/mL

[0303] Titer of WHO Standard for HIV-1 Group M was converted to IU/mL.

[00001]$\mathrm{Titer}\left(\mathrm{in}.Math.\frac{\mathrm{IU}}{\mathrm{mL}}\right)=\frac{\mathrm{Titer}\left(\frac{\mathrm{cp}}{\mathrm{mL}}\right)}{0.6}$

[0304] Therefore HIV-1 Group M LOD in IU/mL is

[0305] LOD by PROBIT analysis (95% hitrate): 6.77 IU/mL

[0306] 95% confidence interval for LOD by PROBIT analysis: 4.75-15.4 IU/mL

TABLE-US-00020 TABLE 2 HIV-1 Group O Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 60 cp/mL 21 21 100%  30 cp/mL 20 20 100%  20 cp/mL 21 21 100%  14 cp/mL 21 19 90%  7 cp/mL 21 15 71% 4.5 cp/mL  21 12 57% 0 cp/mL (neg. control) 12 0  0% LOD by PROBIT analysis (95% hitrate) 14.9 cp/mL 95% confidence interval for LOD by PROBIT 10.9-31.5 analysis cp/mL

[0307] Titer of Primary Standard for HIV-1 Group O was reassigned to CBER HIV-1 Group O panel; calculation factor is 0.586.

[0308] Therefore HIV-1 Group O LOD is

[0309] LOD by PROBIT analysis (95% hitrate): 8.8 cp/mL

[0310] 95% confidence interval for LOD by PROBIT analysis: 6.4-18.5 cp/mL

TABLE-US-00021 TABLE 3 HIV-2 Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 4 cp/mL 21 21 100%  2 cp/mL 21 21 100%  1 cp/mL 21 20 95% 0.5 cp/mL 21 13 62% 0.25 cp/mL 21 13 62% 0.125 cp/mL 21 7 33% 0 cp/mL (neg. control) 12 0  0% LOD by PROBIT analysis (95% hitrate)  1.29 cp/mL 95% confidence interval for LOD by PROBIT −3.11 cp/mL analysis

[0311] Titer of Primary Standard for HIV-2 was reassigned to CBER HIV-2 panel; calculation factor is 26.7.

[0312] Therefore HIV-2 LOD is

[0313] LOD by PROBIT analysis (95% hitrate): 34.44 cp/mL 95% confidence interval for LOD by PROBIT analysis: 21.89-83.04 cp/mL

[0314] HBV

TABLE-US-00022 TABLE 4 HBV Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 7.6 IU/mL 21 21 100%  3.8 IU/mL 21 21 100%  1.9 IU/mL 21 20 95% 0.95 IU/mL  21 14 67% 0.6 IU/mL 19 12 63% 0.4 IU/mL 21 12 57% 0 IU/mL (neg. control) 12 0  0% LOD by PROBIT analysis (95% hitrate) 2.27 IU/mL 95% confidence interval for LOD by PROBIT 1.48-6.54 analysis IU/mL

[0315] HCV

TABLE-US-00023 TABLE 5 HCV Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 24 IU/mL 21 21 100%  12 IU/mL 21 21 100%  6 IU/mL 21 21 100%  3 IU/mL 21 17 81% 1.5 IU/mL 21 14 67% 0.75 IU/mL 21 9 43% 0 IU/mL (neg. control) 18 0  0% LOD by PROBIT analysis (95% hitrate) 4.76 IU/mL 95% confidence interval for LOD by PROBIT 3.14-11.61 analysis IU/mL

[0316] 2. Qualitative analysis of WNV

[0317] Mastermix

TABLE-US-00024 Conc. in Reagent 50 ul-PCR (uM) R1: Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH: 441 R2: DMSO (%) 5.4 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.027 K acetate pH 7.0 120′000  Glycerol (%) 3 Tween 20 (%) 0.015 Tricine pH 8.0 60′000  NTQ21-46A - Aptamer 0.2222 Uracil-N-Glycosylase (U/uL) 0.2 dGTP 400.0 dATP 400.0 dCTP 400.0 dUTP 800.0 ZO5-D Polymerase (U/ul)* 0.9 Primers/probes selected from SEQ ID NOs 53-59 0.08-0.4 SEQ ID NO 42 0.150 SEQ ID NO 43 0.150 SEQ ID NO 44 0.100

[0318] Analytical Sensitivity/LOD

[0319] For the viruses (WNV, SLEV and JEV) an independent panel was prepared as a dilution series of the respective Standard including several concentrations/levels at and around the anticipated LOD. One panel per virus and concentration was tested with at least 20 valid replicates per concentration.

[0320] The LOD was determined by PROBIT analysis.

TABLE-US-00025 TABLE 6 WNV Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 20 cp/mL 21 21 100% 12 cp/mL 21 21 100% 8 cp/mL 21 21 100% 5 cp/mL 21 17  81% 2.5 cp/mL 21 15 71.4%  0.5 cp/mL 21 1  4.8% 0 cp/mL (neg. control) 12 0  0% LOD by PROBIT analysis (95% hitrate) 6.57 cp/mL 95% confidence interval for LOD by PROBIT 4.74-11.03 analysis cp/mL

TABLE-US-00026 TABLE 7 SLEV Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 140 cp/mL  21 21  100% 100 cp/mL  21 20 95.2% 70 cp/mL 21 20 95.2% 40 cp/mL 21 17 81.0% 20 cp/mL 21 11 52.4% 10 cp/mL 21 6 28.6% 0 cp/mL (neg. control) 12 0   0% LOD by PROBIT analysis (95% hitrate) 78.9 cp/mL 95% confidence interval for LOD by PROBIT 55.4-145.7 analysis cp/mL

TABLE-US-00027 TABLE 8 JEV Hit rates and Probit LOD from individual panel Number of Number of Concentration replicates positives Hit rate 20 cp/mL 21 20 95.2% 12 cp/mL 21 20 95.2% 8 cp/mL 21 18 85.7% 5 cp/mL 21 17 81.0% 2.5 cp/mL 21 14 66.7% 0.5 cp/mL 21 2 9.52% 0 cp/mL (neg. control) 12 0   0% LOD by PROBIT analysis (95% hitrate) 13.55 cp/mL 95% confidence interval for LOD by PROBIT 8.78-27.7 analysis cp/mL

[0321] 3. Quantitative analysis of HBV

[0322] Mastermix

TABLE-US-00028 Final Conc. in Reagent 50 ul-PCR (uM) R1: Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic 3′300 Acid) NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH: 6.41 R2: Glycerol (%, w/v)    3% Tricine   60 mM DMSO (%, v/v)  5.4% KOAc  120 mM Tween 20 (v/v) 0.015% Aptamer NTQ21-46 A 0.222 μM   ZO5D Polymerase 0.9 U/μL (45 U/rxn) Uracil-N-Glycosylase 0.2 U/μL (10 U/rxn) Sodium Azide (w/v) 0.027% dCTPs 400 μM dGTPs 400 μM dATPs 400 μM dUTPs 800 μM SEQ ID NO 36  1.2 μM SEQ ID NO 37  1.2 μM SEQ ID NO 50  0.6 μM SEQ ID NO 51  0.6 μM SEQ ID NO 38  0.1 μM SEQ ID NO 52 M

[0323] Analytical Sensitivity/LOD

[0324] Four dilution panels were prepared with HBV Secondary Standard (representing Genotype A), i.e., two in HBV negative serum for sample input volumes of 200 μL and 500 μL, and two in HBV negative EDTA-plasma for sample input volumes of 200 μL and 500 μL. Each panel included 7 concentration levels at and around the anticipated LOD. One panel per matrix was tested with ≧21 replicates per concentration level. At least 20 replicates needed to be valid. The LOD was determined by PROBIT analysis at 95% hit rate and by ≧95% hit rate analysis.

TABLE-US-00029 TABLE 9 LOD analysis for 200 μL input volume in EDTA-plasma. * Number of Number of Concentration replicates positives Hit rate 25 IU/mL 41 41  100% 15 IU/mL 41 39 95.1% 10 IU/mL 41 40 97.6%  7 IU/mL 41 40 97.6%  4 IU/mL 24 20 83.3%  1 IU/mL 24 4 16.7% 0 IU/mL (neg. control) 24 0   0% LOD by PROBIT analysis (95% hitrate) 8.2 IU/mL 95% confidence interval for LOD by PROBIT 4.8-26.0 analysis IU/mL * Additional replicates were tested to narrow the observed 95% confidence interval.

TABLE-US-00030 TABLE 10 LOD analysis for 500 μL input volume in EDTA-plasma. Number of Number of Concentration replicates positives Hit rate 10 IU/mL 21 21 100% 7 IU/mL 21 21 100% 4 IU/mL 21 21 100% 2.5 IU/mL 21 20 95.2%  1 IU/mL 21 14 66.7%  0.2 IU/mL 21 1  4.8% 0 IU/mL (neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 2.3 IU/mL 95% confidence interval for LOD by PROBIT 1.6-4.2 analysis IU/mL

TABLE-US-00031 TABLE 11 LOD analysis for 200 μL input volume in serum. Number of Number of Concentration replicates positives Hit rate 25 IU/mL 21 21  100% 15 IU/mL 21 20 95.2% 10 IU/mL 21 21  100% 7 IU/mL 21 20 95.2% 4 IU/mL 21 15 71.4% 1 IU/mL 21 8 38.1% 0 IU/mL (neg. control) 21 0   0% LOD by PROBIT analysis (95% hitrate) 9.4 IU/mL 95% confidence interval for LOD by PROBIT 6.2-19.0 analysis IU/mL

TABLE-US-00032 TABLE 12 LOD analysis for 500 μL input volume in serum. Number of Number of Concentration replicates positives Hit rate 10 IU/mL 21 21 100% 7 IU/mL 21 21 100% 4 IU/mL 21 21 100% 2.5 IU/mL 21 16 76.2%  1 IU/mL 21 16 76.2%  0.2 IU/mL 21 7 33.3%  0 IU/mL (neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 4.1 IU/mL 95% confidence interval for LOD by PROBIT 2.4-10.0 analysis IU/mL

[0325] Summary LOD:

[0326] EDTA-plasma: The PROBIT analysis at 95% hit rate resulted in an LOD of 8.2 IU/mL for 200 μL sample input volume and 2.3 IU/mL for 500 μL sample input volume for EDTA-plasma.

[0327] The 95% confidence interval range for these concentrations was 4.8-26.0 IU/mL for 200 μL sample input volume and 1.6-4.2 IU/mL for 500 μL sample input volume.

[0328] Serum: The PROBIT analysis at 95% hit rate resulted in an LOD of 9.02 IU/mL for 200 μL sample input volume and 4.1 IU/mL for 500 μL sample input volume for serum.

[0329] The 95% confidence interval range for these concentrations was 6.2-19.0 IU/mL for 200 μL sample input volume and 2.4-10.0 IU/mL for 500 μL sample input volume.

[0330] Linearity

[0331] One EDTA-plasma panel and one serum panel were prepared by using HBV genotype A (provided by RMD Research Pleasanton, linearized plasmid, pHBV-PC_ADW2). Each of the panels was analyzed at 12 concentration levels for the determination of the expected dynamic range (4-2E+09 IU/mL) of the assay. All concentration levels/panel members (PM) were tested in 21 replicates.

[0332] This study was done with a sample input volume of 500 μL. The concentration levels were selected as follows: One level below expected Lower Limit of Quantitation (LLOQ), one at expected LLOQ, one above expected LLOQ, several concentrations at intermediates levels, at expected Upper Limit of Quantitation (ULOQ) and one above expected ULOQ.:

[0333] PM 12—2.0E+09 IU/mL—above expected ULOQ

[0334] PM 11—1.0E+09 IU/mL—at expected ULOQ

[0335] PM 10—1.0E+08 IU/mL—below expected ULOQ

[0336] PM 9—1.0E+07 IU/mL—intermediate concentration level

[0337] PM 8—1.0E+06 IU/mL—intermediate concentration level

[0338] PM 7—1.0E+05 IU/mL—intermediate concentration level

[0339] PM 6—1.0E+04 IU/mL—intermediate concentration level

[0340] PM 5—1.0E+03 IU/mL—intermediate concentration level

[0341] PM 6a—2.0E+02 IU/mL—intermediate concentration level (PM 6 diluted to 2.0E+02 IU/mL, used for titer assignment of serum panel)

[0342] PM 4—1.0E+02 IU/mL—intermediate concentration level (also used for titer assignment of plasma panel)

[0343] PM 3—5.0E+01 IU/mL—above expected LLOQ

[0344] PM 2—1.0E+01 IU/mL—at expected LLOQ

[0345] PM 1—4.0E+00 IU/mL below expected LLOQ

[0346] For every valid sample of the linearity panel, the observed HBV DNA titer was transformed to log10 titer and the mean log10 titer was calculated per concentration level.

TABLE-US-00033 TABLE 13 Linearity in EDTA Plasma Assigned Nominal Titer Assigned Titer Log10 Mean Log 10 (IU/mL) (IU/mL) Titer Titer observed Replicates 4.00E+00 3.50E+00 0.54 0.52 17 1.00E+01 8.70E+00 0.94 0.91 21 5.00E+01 4.40E+01 1.64 1.69 21 1.00E+02 8.70E+01 1.94 2.04 21 1.00E+03 8.70E+02 2.94 3.01 21 1.00E+04 8.70E+03 3.94 3.9 21 1.00E+05 8.70E+04 4.94 4.88 21 1.00E+06 8.70E+05 5.94 5.87 21 1.00E+07 8.70E+06 6.94 6.92 21 1.00E+08 8.70E+07 7.94 8.01 21 1.00E+09 8.70E+08 8.94 9.04 21 2.00E+09 1.70E+09 9.24 9.38 21

[0347] A graphical depiction of this result is shown in FIG. 3.

TABLE-US-00034 TABLE 14 Linearity in Serum Assigned Nominal Titer Assigned Titer Log10 Mean Log 10 (IU/mL) (IU/mL) Titer Titer observed Replicates 4.00E+00 3.30E+00 0.52 0.7 21 1.00E+01 8.30E+00 0.92 0.99 21 5.00E+01 4.10E+01 1.62 1.73 21 1.00E+02 8.30E+01 1.92 2.03 21 1.00E+03 8.30E+02 2.92 2.93 21 1.00E+04 8.30E+03 3.92 3.8 21 1.00E+05 8.30E+04 4.92 4.78 21 1.00E+06 8.30E+05 5.92 5.75 21 1.00E+07 8.30E+06 6.92 6.73 21 1.00E+08 8.30E+07 7.92 7.78 21 1.00E+09 8.30E+08 8.92 8.92 21 2.00E+09 1.70E+09 9.22 9.22 21

[0348] A graphical depiction of this result is shown in FIG. 4.

[0349] Summary Linearity:

[0350] The linear range, defined as the concentration range for which the log10 deviation of the mean log10 observed titers is within ±0.3 of the log10 nominal titer was determined as: 3.5E+00 IU/mL-1.7E+09 IU/mL for EDTA-plasma and 3.3E+00 IU/mL-1.7E+09 IU/mL for serum. The Lower Limit of Quantitation was found to be: 4.0E+00 IU/mL for EDTA-plasma and serum.

[0351] 4. Quantitative analysis of HCV

[0352] Mastermix

TABLE-US-00035 R1: Final Conc. in Reagent 50 ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH: 6.41 R2: Final Conc. in Reagent 50 ul-PCR Glycerol (%, w/v)    3% Tricine   60 mM DMSO (%, v/v)  5.4% KOAc  120 mM Tween 20 (v/v) 0.015% NTQ21-46 A 0.222 μM   ZO5D 0.9 U/μL (45 U/rxn) UNG 0.2 U/μL (10 U/rxn) Sodium Azide (w/v) 0.027 dCTPs 400 μM dGTPs 400 μM dATPs 400 μM dUTPs 800 μM Primers/probes selected from SEQ ID NOs 60-76  0.1 μM SEQ ID NO 42  0.3 μM SEQ ID NO 43  0.3 μM SEQ ID NO 44 μM

[0353] Analytical Sensitivity/LOD

[0354] A dilution panel was prepared with Roche HCV Secondary Standard in HCV negative EDTA plasma and serum using a sample input volumes of 200 μL and 500 μL. Each concentration level was tested with 21 replicates. At least ≧20 replicates have to be valid. The LOD was determined by PROBIT analysis at 95% hit rate and by ≧95% hit rate analysis.

TABLE-US-00036 TABLE 15 Hit rates and Probit with 200 μL sample process input volume for EDTA-plasma Number of Number of Concentration replicates positives Hit rate 55 IU/mL 21 21 100%  38 IU/mL 21 21 100%  25 IU/mL 21 20 95% 12.5 IU/mL 21 19 90% 6 IU/mL 21 15 71% 3 IU/mL 21 6 29% 0 IU/mL (neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 17.4 IU/mL 95% confidence interval for LOD by PROBIT 12.1-34.3 analysis IU/mL

TABLE-US-00037 TABLE 16 Hit rates and Probit with 500 μL sample process input volume for EDTA-plasma Number of Number of Concentration replicates positives Hit rate 22 IU/mL 21 21 100%  15 IU/mL 21 21 100%  10 IU/mL 20 20 100%  5 IU/mL 21 19 76% 2.5 IU/mL 21 15 71% 1 IU/mL 21 6 57% 0 IU/mL (neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 9.0 IU/mL 95% confidence interval for LOD by PROBIT 5.5-25.4 analysis IU/mL

TABLE-US-00038 TABLE 17 Hit rates and Probit with 200 μL sample process input volume for serum Number of Number of Concentration replicates positives Hit rate 55 IU/mL 21 21 100%  38 IU/mL 21 21 100%  25 IU/mL 21 20 95% 12.5 IU/mL 21 18 86% 6 IU/mL 21 13 62% 3 IU/mL 21 6 29% 0 IU/mL (neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 20.2 IU/mL 95% confidence interval for LOD by PROBIT 14.0-39.3 analysis IU/mL

TABLE-US-00039 TABLE 18 Hit rates and Probit with 500 μL sample process input volume for serum Number of Number of Concentration replicates positives Hit rate 22 IU/mL 21 21 100%  15 IU/mL 21 21 100%  10 IU/mL 21 20 95% 5 IU/mL 21 18 86% 2.5 IU/mL 21 12 57% 1 IU/mL 21 4 19% 0 IU/mL (neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 8.2 IU/mL 95% confidence interval for LOD by PROBIT 5.8-15.0 analysis IU/mL

[0355] Summary LOD:

[0356] 1. The PROBIT analysis at 95% Hit rate resulted in an LOD of 17.4 IU/mL for 200 μL sample process input volume and 9.0 IU/mL for 500 μL sample process input volume for EDTA plasma. The 95% confidence interval for these concentrations is 12.1-34.3 IU/mL for 200 μL sample process input volume and 5.5-25.4 IU/mL for 500 μL sample process input volume.

[0357] 2. The values of the PROBIT analysis at 95% Hit rate is 20.2 IU/mL for 200 μL sample process input volume and 8.2 IU/mL for 500 μL sample process input volume for serum. The 95% confidence interval for these concentrations is 14.0-39.3 IU/mL for 200 μL sample process input volume and 5.8-15.0 IU/mL for 500 μL sample process input volume.

[0358] Linearity

[0359] One preparation of an EDTA-plasma panel and one preparation of a serum panel of HCV aRNA traceable to the HCV WHO Standard were analyzed. The linearity panels were prepared by serial dilution and analyzed at 10 different concentrations. The study was done with 500 μL sample process input volume. The concentrations were selected as follows: One level below expected Lower Limit of Quantification (LLoQ), one at LLoQ, one above LLOQ, several concentrations at intermediates levels, at expected Upper Limit of Quantification (ULoQ) and one at or above ULoQ.

[0360] For all concentrations 21 replicates were tested.

[0361] PM 1—2.0E+08 IU/mL—above expected ULoQ

[0362] PM 2—1.0E+08 IU/mL—at expected ULoQ

[0363] PM 3—1.0E+07 IU/mL—below expected ULoQ

[0364] PM 4—1.0E+06 IU/mL—intermediate concentration level

[0365] PM 5 1.0E+05 IU/mL—intermediate concentration level

[0366] PM 6—1.0E+04 IU/mL—intermediate concentration level for titer assignment

[0367] PM 7—1.0E+03 IU/mL—intermediate concentration level

[0368] PM 8—1.0E+02 IU/mL—above expected LLoQ

[0369] PM 9—1.0E+01 IU/mL—at expected LLoQ

[0370] PM 10—8.0E+00 IU/mL—below expected LLoQ

TABLE-US-00040 TABLE 19 Linearity in EDTA Plasma Assigned Nominal Titer Assigned Titer Log10 Mean Log 10 (IU/mL) (IU/mL) Titer Titer observed Replicates 8.00E+00 4.87E+00 0.7 0.6 15 1.00E+01 6.09E+00 0.8 0.8 17 1.00E+02 6.09E+01 1.8 1.7 21 1.00E+03 6.09E+02 2.8 2.8 21 1.00E+04 6.09E+03 3.8 3.8 21 1.00E+05 6.09E+04 4.8 4.7 21/20 1.00E+06 6.09E+05 5.8 5.6 21/20 1.00E+07 6.09E+06 6.8 6.7 21 1.00E+08 6.09E+07 7.8 7.8/7.7 21/18 2.00E+08 1.22E+08 8.1 8   21/20

[0371] A graphical depiction of this result is shown in FIG. 5.

TABLE-US-00041 TABLE 20 Linearity in Serum Assigned Nominal Titer Assigned Titer Log10 Mean Log 10 (IU/mL) (IU/mL) Titer Titer observed Replicates 8.00E+00 3.90E+00 0.6 0.7 10 1.00E+01 4.96E+00 0.7 0.7 14 1.00E+02 4.96E+01 1.7 1.6 21 1.00E+03 4.96E+02 2.7 2.8 21 1.00E+04 4.96E+03 3.7 3.7 21 1.00E+05 4.96E+04 4.7 4.7 21 1.00E+06 4.96E+05 5.7 5.7 21 1.00E+07 4.96E+06 6.7 6.7 21 1.00E+08 4.96E+07 7.7 7.7 21 2.00E+08 9.92E+07 8 8.1 21

[0372] A graphical depiction of this result is shown in FIG. 6.

[0373] Summary Linearity:

[0374] The linear range, defined as the concentration range for which the log10 deviation of the mean log10 observed titers is within ±0.3 of the log10 nominal titer was determined as: 4.87E+00 IU/mL-1.22E+08 IU/mL for EDTA-plasma and 3.90E+00 IU/mL-9.92E+07 IU/mL for serum.

[0375] 5. Quantitative analysis of HIV

TABLE-US-00042 Mastermix R1: Final Conc. in Reagent 50 ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH: 6.41 R2: Final Conc. in Reagent 50 ul-PCR Glycerol (%, w/v)   3% Tricine   60 mM DMSO (%, v/v)  5.4% KOAc  120 mM Tween 20 (v/v) 0.02% Aptamer NTQ21-46 A 0.222 μM   ZO5D Polymerase 0.9 U/μL (45 U/rxn) UNG 0.2 U/μL (10 U/rxn) Sodium Azide (w/v) 0.027 dCTPs 400 μM dGTPs 400 μM dATPs 400 μiM dUTPs 800 μM Primers/probes selected from SEQ ID NOs 1-35 0.1 μM-0.3 μM SEQ ID NO 50  0.3 μM SEQ ID NO 51  0.3 μM SEQ ID NO 52 μM

[0376] Analytical Sensitivity/LOD

[0377] A dilution panel was prepared with HIV-1M Secondary Standard in HIV-1 negative EDTA plasma for sample input volumes of 200 μL and 500 μL. Each concentration level was tested with 21 replicates. At least ≧20 replicates have to be valid. The LOD was determined by PROBIT analysis at 95% hit rate and by ≧95% hit rate analysis.

TABLE-US-00043 TABLE 21 LOD analysis for 200 μL input volume in EDTA-plasma Number of Number of Concentration replicates positives Hit rate 200 cp/mL  21 21  100% 100 cp/mL  21 21  100% 80 cp/mL 21 21  100% 50 cp/mL 21 20 95.2% 30 cp/mL 21 18 85.7% 20 cp/mL 21 17 81.0% 10 cp/mL 21 8 38.1% 0 cp/mL (neg. control) 21 0   0% LOD by PROBIT analysis (95% hitrate) 41.8 cp/mL 95% confidence interval for LOD by PROBIT 30.9-74.9 analysis cp/mL

TABLE-US-00044 TABLE 22 LOD analysis for 500 μL input volume Number of Number of Concentration replicates positives Hit rate 30 cp/mL 21 21  100% 25 cp/mL 21 20 95.2% 20 cp/mL 21 21  100% 13.5 cp/mL 21 18 85.7% 9 cp/mL 21 13 61.9% 6 cp/mL 21 9 42.9% 0 cp /mL (neg. control) 21 0   0% LOD by PROBIT analysis (95% hitrate) 18.9 cp/mL 95% confidence interval for LOD by PROBIT 14.9-29.4 analysis cp/mL

[0378] Summary LOD

[0379] 1. The PROBIT analysis at 95% hit rate resulted in an LOD of 41.8 cp/mL for 200 μL input volume and 18.9 cp/mL for 500 μL input volume.

[0380] 2. The 95% confidence interval range for these concentrations was 30.9-74.9 cp/mL for 200 μL input volume and 14.9-29.4 cp/mL for 500 μL input volume.

[0381] Linearity

[0382] The samples used in the Linearity/Dynamic Range/Accuracy study consisted of a dilution panel of an HIV-1 cell culture supernatant material, HIV-1 group M subtype B.

[0383] The linearity panel was prepared by serial dilution. This panel was analyzed at 10 concentration levels.

[0384] The concentrations were selected as follows: One level below expected Lower Limit of Quantitation (LLoQ), one at LLoQ, one above LLoQ, several concentrations at intermediate levels, at expected Upper Limit of Quantitation (ULoQ) and one above ULoQ. For all concentrations 21 replicates were tested. The linearity study was done with 500 μL input volume):

[0385] PM 1—2.0E+07 cp/mL—above expected ULoQ

[0386] PM 2—1.0E+07 cp/mL—at expected ULoQ

[0387] PM 3—1.0E+06 cp/mL—below expected ULoQ

[0388] PM 4—1.0E+05 cp/mL—intermediate concentration level

[0389] PM 5 3.0E+04 cp/mL—intermediate concentration level for titer assignment

[0390] PM 6—1.0E+04 cp/mL—intermediate concentration level

[0391] PM 7—1.0E+03 cp/mL—intermediate concentration level

[0392] PM 8—1.0E+02 cp/mL—intermediate concentration level

[0393] PM 9—5.0E+01 cp/mL—above expected LLoQ

[0394] PM 10—2.0E+01 cp/mL—at expected LLoQ

[0395] PM 11—1.5E+01 cp/mL below expected LLoQ

TABLE-US-00045 TABLE 23 Linearity in EDTA Plasma Assigned Nominal Titer Assigned Titer Log10 Mean Log 10 (cp/mL) (cp/mL) Titer Titer observed Replicates 1.50E+01 1.50E+01 1.2 1.3 21 2.00E+01 2.00E+01 1.3 1.5 21 5.00E+01 5.10E+01 1.7 1.8 21 1.00E+02 1.00E+02 2 2 21 1.00E+03 1.00E+03 3 3 21 1.00E+04 1.00E+04 4 4 21 1.00E+05 1.00E+05 5 5 21 1.00E+06 1.00E+06 6 6 21 1.00E+07 1.00E+07 7 7 21 2.00E+07 2.00E+07 7.3 7.4 21

[0396] A graphical depiction of this result is shown in FIG. 7.

[0397] Summary Linearity

[0398] The linear range, defined as the concentration range for which the log10 deviation of the mean log10 observed titers is within ±0.3 of the log10 nominal titer was determined as 1.5E+01 cp/mL-2.0E+07 cp/mL.

Example 3

[0399] Using the generic amplification process described hereinabove for the amplification of nucleic acids (both RNA and DNA) can lead to over quantification if the desired target nucleic acid is DNA but there is a significant amount of RNA present such that amplification of the undesired RNA can occur. One method to eliminate the undesired amplification of RNA is to employ RNase H which degrades RNA that is present in a RNA-DNA hybrid complex. An experiment was therefore performed for the quantitative analysis of HBV as described in Example 2, Section 3 with the exception of the addition in the R2 Mastermix reagent of 1 unit (per 50 μl PCR reaction) of either Hybridase™ Thermostable RNase H (Epicentre, Madison, Wis.) or recombinant E. Coli RNase H (New England Biolabs, Ipswich, Mass.). Amplification was performed on an RNA template (between 30,000 and 3,000,000 copies per reaction) or on an DNA template (between 30 and 3000 copies per reaction). The results of the experiment are shown on Table 24. Reactions with the DNA template in the presence of either RNase H enzyme showed no difference in amplification efficiency compared to reactions in the absence of RNase H. In contrast, the amplification of the RNA template was severely delayed or even absent (for Hybridase™), clearly showing that the RNA template was degraded by the addition of the RNase H enzyme prior to amplification.

TABLE-US-00046 TABLE 24 Effect of RNase H on RNA and DNA amplification Copy number 3.0E+06 3.0E+05 3.0E+04 3.0E+03 3.0E+02 3.0E+01 RNA 21.8 25.5 28.6 Ave. Cp RNA + Rnase H 28.8 32.3 38.0 (NEB) Ave. Cp RNA + Rnase H 36.9 >50 (Neg) >50 (Neg) (Epi) Ave. Cp DNA 23.6 27.0 30.4 Ave. Cp DNA + Rnase H 23.5 26.9 30.5 (NEB) Ave. Cp DNA + Rnase H 23.4 27.0 30.6 (Epi) Ave. Cp

[0400] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.