Detection Methods For Viralload Assessment

A number of assays are currently commercially available for the analysis of viral loads and are used in clinical laboratories. These include RT-PCR (Amplicor-Roche), bDNA (Chiron), and molecular hybridization (Digene), although the FDA has only approved RT-PCR for HIV viral-load assessment, These assays are able to detect as few as 50 copies/mL in serum and represent an important advance in the diagnosis and monitoring viral disease. Since most of these assays rely on amplification, variability is introduced and CVs are often high. As such, advances in technology, i.e., CE-LIF, that improve reliability, quantitation, and detection are needed. CE-LIF detection that is able to detect attomolar concentrations is highly reproducible and fully automatable has the potential for improving viral-load analysis.

4.1. Slab Gel Electrophoresis

PCR products are commonly analyzed by slab gel electrophoresis (SGE) (see Fig. 6), where an agarose gel containing ethidium bromide acts as the separation medium and products are visualized directly with UV light. Visualization by SGE is a rapid and inexpensive method for the analysis of PCR products. The major limitation of SGE is a relatively high detection limit, usually on the order of nanograms. Slab gel electrophoresis with radiolabeled probes is able to detect 1-5 pg of target DNA with an overnight exposure. Radioactive-detection methods, however, have significant disadvantages with respect to safety, stability of the labeled nucleic acids, and automation (29).

4.2. Capillary Electrophoresis with Laser-Induced Fluorescence

High pressure liquid chromatography (HPLC) was initially studied as a replacement for slab gel electrophoresis, although restricted intraparticle diffusion of biopolymers resulted in only limited improvement of resolution and speed (30,31). Alternatively, electrophoretic separations (CE) can be performed in narrow-bore tubes or capillaries and may be viewed simply as another mode of electrophoresis (32-34). Originally, CE utilizing hydroxyethylcellulose and ethidium bromide was shown to have increased resolving power when compared to HPLC for dsDNA, however, the detection level still remained in the nanogram range.

CE has since been used successfully in the research setting to separate and quantitate PCR products of HIV-1, HBV, CMV, and HCV (35-39). The introduction of CE with LIF and intercalating dyes improved detectability to the attomole level with sample volumes as little as a few picoliters (4043). CE represents a safe and automatable assay system for quantitative analysis and routine laboratory analysis may soon be the norm.

Capillary gel electrophoresis (CGE) initially used either a fixed or immobilized polymerized matrix which acts as a "molecular sieve" within the capillary to separate DNA based on size and charge (44-46). Since the mass to charge ratio of DNA remains constant with increasing mass, separations are made based on differences in molecular weight. As charged solutes migrate through the polymer network, they are retarded with larger molecules being retarded, more than smaller ones, allowing for separation based on molecular weight. Initially, CE used crosslinked agarose and polyacrylamide for the separation of DNA. However, polymerization of gels within the capillary is difficult and time-consuming. Polymerization that occurs too rapidly, use of impure chemicals and solutions that are not degassed can lead to bubble formation and unstable gels. Additionally, these capillaries are very rigid, making hydrodynamic injections impossible and the capillary susceptible to breakage. Linear polymer solutions are more flexible and pressure can be used to refill the capillary (47,48). Additionally, they are much less susceptible to bubble formation and breakage. Although the polymer structure of the cross-linked gel is much different than the linear polymer solution, the mechanism of separation is identical and the ease of use with the replaceable polymer networks have made these the capillary system of choice for most laboratories (49,50). CGE is used most frequently for the analysis of nucleic acids and will be the focus of this discussion.

4.2.1. CE-LIF Analysis of RT-PCR Products

To quantitate viral load, the measured concentration must be a reliable gauge of the amount of nucleic acid present in the original sample. This is usually a two-part process; amplification followed by detection. RNA

Fig. 7. Electropherogram of PCR products analyzed by CE-LIF. RNA obtained from MCF-7 cells is amplified by RT-PCR and analyzed. DNA elutes at 21 min, indicating the presence of the target gene in the sample.

samples are usually amplified by RT-PCR. In this process an internal standard that will amplify under the same conditions as the target sequence is introduced prior to amplification. The internal standard is used to control for variability within the PCR reaction as well as to provide a reference to calculate the initial concentration of unknown (51) (see Fig. 7).

To ensure peak areas reported by CE-LIF are an accurate measure of the amount of RT-PCR product present in the sample, a calibration curve is generated by injecting known concentrations DNA. The same solution of standard DNA fragments can also be used to calibrate the capillary and determine the molecular weight of fragments based on retention times. In addition to being an additional step towards the accurate quantitation of gene expression, this technique offers several advantages. Sample volumes are small (1-5 ^L), sensitivity is high (attomolar), and the hazards associated with isotopic storage, use and disposal are eliminated. This technique can also be automated, used for fraction collection, and validated. There are, however, drawbacks to CE-LIF, including expense. Initially, there is an investment in equipment, CE, laser, computer and application software that must be made. In addition, consumable supplies, primarily intercalating dyes and capillaries can also be expensive and in the case of capillaries, fragile.

With current CE-LIF technology, samples are analyzed individually (52). Each sample usually has a 15-45 min run time, whereas multiple samples can be analyzed simultaneously by SGE. For analysis of a single sample, CE-LIF may be faster, but when multiple analyses are required, SGE may be more time efficient. With the multicapillary instruments currently under development, analysis of multiple samples simultaneously by CE-LIF will be possible. In addition to requiring a longer time, individual sample processing also means that each sample is analyzed separately increasing risk of bias. Of the steps involved in analyzing PCR products, injection bias is the most frequent source of error, although this can be minimized by using hydrodynamic injections. Interassay variation can also be minimized by the addition of a reference of known concentration into each of the samples after PCR amplification. DNA fragments of similar, but not identical molecular weight to the unknown sample are best used as a reference. Although not currently used for commercial analysis of viral loads, CE-LIF represents an important avenue for exploration. CE-LIF has been used to analyze HIV, HBV, HCV, and CMV, in addition to many other PCR-gener-ated fragments (53,54). Experimental Conditions and Reagents: RNA Purification Total cellular RNA and RNA obtained from human tissue samples including tumor biopsies and whole blood can be obtained by standard procedures. Alternatively, commercially available system such as Ultraspec II RNA isolation system (Biotecx, Houston, TX) may be used. This system isolates total RNA by disruption and homogenization of samples with 14 M guanidine salts and urea followed by chloroform extraction. The sample is centrifuged and the upper aqueous phase containing the RNA is isolated followed by isopropanol precipitation. A proprietary RNATack resin that specifically binds RNA and then eluted with TE (Tris-EDTA, pH 7.4) buffer purifies the RNA. The RNA concentration is quantitated spectrophotometri-cally. The entire isolation can be completed in approx 1 h, which is a significant advantage over standard methods. RNA can also be extracted directly from lymphocytes obtained from whole blood. Concentrations of 10-15 ng of RNA are routinely obtained from 10 mL of whole blood comparing favorably to standard methods (55). Experimental Conditions and Reagents: Design of Internal Standard An internal standard may be designed by purifying, using SGE, the desired PCR product and identifying restriction sites 30-70 base pairs apart that will generate compatible ends. Digestion with the appropriate enzymes and re-ligation generates a DNA fragment that is identical to the target DNA, but 30-70 base pairs smaller. The primer recognition sites and the sequence are identical and the internal standard should amplify under identical conditions. After gel purification and elution in TE, the internal standard is quantified spectrophotometrically and stored at -20°C. The internal standard concentration is then titrated to determine what concentration is optimal for amplification, usually 10-6 ng/PCR reaction.

Using a DNA standard has the benefit of ease of preparation and storage, amplification under identical conditions, and low expense. It does not, however, control for variability present within the RT step. Thus, RNA standards are usually introduced prior to reverse transcription to control for variability throughout the RT-PCR process (51). Experimental Conditions and Reagents: RT-PCR

To improve reproducibility, all RT and PCR steps are done with master mixes that contain all components except the target nucleotides and Taq polymerase. Since PCR is by nature prone to contamination and false-positive results, precautions must be taken to ensure the validity of results. All reagents should be aliquotted into single use portions and separate pipets should be set aside to be used only for PCR. Reactions can be set-up in a biological hood and all surfaces exposed to UV light between reactions. Adequate controls (both positive and negative) should be used for all reactions (27). Experimental Conditions and Reagents: Instrument Parameters

Separations are performed on a CE-LIF system, with the temperature held constant at 20°C. PCR products are detected by LIF in the reversed polarity mode (anode at the detector site) with excitation at 488 nm and emission at 520 nm. Samples are introduced hydrodynamically using 10-s injections at 0.5 psi into a 100 mm i.d. x 65 cm coated (neutral) capillary filled with TBE containing replaceable linear polyacrylamide. No sample preparation of PCR products is required. The capillary is conditioned with buffer containing 60 ^g thiazole orange (an intercalator) per 20 mL and rinsed at high pressure for 3 min. Separations are performed under constant voltage at 7.09.0 kV for 15-50 min. The capillary is rinsed with gel buffer for 3 min prior to each injection (51).

4.2.2. Direct Detection of Nucleic Acids

Reliance on an amplification step is the major problem associated with PCR based methodology (56). Despite the incorporation of internal standards, quantitation is still problematic, particularly when the target template is small. To decrease the variability associated with PCR-based assays and to take advantage of the exquisite sensitivity of CE-LIF, the direct detection of nucleic acids has been developed.

HIV RNA/P robe Complex

Fig. 8. Electropherogram analysis of hybridization products. RNA samples obtained from a HIV seropositive patient were hybridized with a HIV specific probe and analyzed. HIV RNA/Probe complex elutes at 12 min, indicating the presence of HIV RNA in the patient's serum.

A report has attempted to quantify HIV-1 RNA directly from the plasma by assuming that all plasma RNA is due to HIV-1. However, without specific HIV-1 probes, plasma samples may contain non-HIV-1 viral RNA including HTLV-1 (human T-cell leukemia virus Type-1), and hepatitis A, C, D, and E, which reduces the specificity for HIV-1. Additionally, contamination of plasma with leukocytes or other cells would result in the presence of nonspecific human RNA.

In an alternative approach, cellular RNA is hybridized with a HIV-1 specific probe that has been labeled with fluorescene. A complex is formed if HIV-1 RNA is present and unbound RNA is digested with RNAase I. Samples are then analyzed by CE-LIF with thiazole orange or other intercalators present in the buffer system. Two peaks elute if HIV-1 RNA is present; the first is the DNA/DNA unbound probe complex, followed by the DNA/RNA hybrid. Although the complexes are the same lengths, the DNA/ RNA complex has a different secondary structure and slightly higher molecular weight and a subsequently longer retention time (see Fig. 8).

Thiazole orange present in the buffer intercalates into 1 out of 2 bp for DNA and 1 out of 10 bp for RNA. Although the intercalation parameters of thiazole orange into a DNA/RNA complex is unknown, it is assumed to be between 10 and 50%, providing a 10-50% enhancement in sensitivity over the RNA/RNA complex. The addition of a fluorescein label to the probe provides a double-detection system over the intercalator alone, also enhancing sensitivity.

The double-detection system is linear from 0.072-21.46 pg, the migration time precision is <1% and the peak-area precision ranges from 1-11%. The minimal detectable level is 36 atg, which corresponds to 4 equivalents (4 copies/mL) of HIV. As little as 19 fg (1710 copies per 1 mL of starting plasma) of HIV-RNA can be reliably and quantitatively detected. Although still a research tool, direct detection of nucleic acids may be an important improvement in assay reliability.

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