Discussion

PCR, with its extraordinary sensitivity, is the method of choice for the detection of nucleic acids present in very low concentrations in biological specimens. Diagnostic applications of this technique mainly concern analysis of gene amplification, expression in tumors, and expression of etiological agents in human infections. Assays that combine PCR amplification and the immunologi-cal chemiluminescent detection of PCR products (PCR-CLEIA) provide high specificity, sensitivity, objectivity, and rapidity. In particular, the use of chemiluminescence as a detection system provides superior assay performance. Chemiluminescence is a versatile analytical tool that offers several advantages over other detection principles, including high detectability, high selectivity, wide dynamic range, and rapidity.[1'2] Thanks to the high sensitivity of chemiluminescence detection, very small volumes of reagents and samples can be used, which results in lower assay costs and the possibility of exploiting high-density formats such as 384-well and 1536-well microtiter plates. This last feature, combined with rapidity, renders chemiluminescence detection suitable for the development of high-throughput methods.

PCR-CLEIA can be very useful in understanding the progression of the disease, monitoring the success of therapy, and evaluating the potential risks of transmission of pathogens. Moreover, PCR-CLEIA can be very useful in the diagnosis of infectious diseases that can persist in the presence of a small number of infectious agents, and in distinguishing low-level innocuous infections from those which may be of clinical relevance.

PCR-CLEIA is based on the following:

1. Direct incorporation of a labeled nucleotide during PCR amplification reaction [mainly by incorporation of digoxigenin (Dig)-labeled dUTP].

2. Hybridization of labeled amplified products with biotin-labeled probes specific for the target (alternatively, the PCR product can be biotin-labeled by performing the amplification reaction in the presence of a biotin-labeled dUTP or primer; in this case, hybridization will be performed with either a Dig-labeled or an enzyme-labeled probe).

3. Capture of hybridized amplicons onto streptavidin-coated white or black microtiter plates (alternatively, the probe can be immobilized onto the solid phase, then the labeled amplicon can be added and hybridization can be performed directly into the wells of the microtiter plate).

4. Detection of immobilized hybridized amplicons by anti-Dig antibodies conjugated to peroxidase or alkaline phosphatase (this step is not necessary in case an enzyme-labeled probe is used).

5. Addition of chemiluminescent substrates with high sensitivity and wide dynamic range.

6. Measurement of chemiluminescent signals by microplate luminometers or imaging systems and correlation to amounts of amplified products.

A schematic representation of the principle of the method is shown in Fig. 1.

With PCR-CLEIA, semiquantitative information on target DNA or RNA is obtained. Moreover, results evaluation is objective, whereas conventional PCR requires a more subjective interpretation; at low DNA concentration, interpretation of results may be doubtful. Unlike conventional PCR, PCR-CLEIA is automatable, able to process simultaneously a large number of samples in reasonable time, and can be applied to DNA or RNA quantification. The chemiluminescent substrate used in amplicon detection permits a large-scale range of measurements compared with colorimetric or fluoro-metric detection.

To develop a standardized PCR-CLEIA, different steps have to be optimized.

Optimization of PCR-CLEIA Method

Streptavidin-coated microtiter plates able to efficiently adsorb biotinylated nucleic acids are commercially available. To exclude high background luminescence emitted by plate formats, preliminary measurement of phosphorescence emission intensity after exposure to ambient light of the microtiter plate can be performed.

Enzyme-labeled anti-Dig Ab

Dig-labeled amplicon

Immobilized biotinylated probe Streptavidin

Fig. 1 Microtiter plate hybridization and immunodetection scheme. Schematic representation of the microtiter plate immunochemiluminescent hybridization assay of PCR products: the Dig-labeled PCR product is hybridized with biotin-labeled specific oligoprobes; hybridized amplicons are captured onto streptavidin-coated microtiter plates, then detected by means of anti-Dig antibodies and chemiluminescent detection. CL, chemiluminescent; Dig, digoxigenin; Ab, antibody.

The optimal concentration of target-specific probe has to be determined. PCR products, obtained from serial dilutions of the original sample, have to be hybridized with different concentrations of probe and, subsequently, captured onto the microtiter plate. The chemiluminescent signal intensity from each well is then measured. The oligoprobe concentration that allows to obtain the calibration curve with the lowest limit of detection and the highest sensitivity (slope of the curve) has to be chosen. Usually, no further improvement is obtained by increasing the oligoprobe concentration.

Direct incorporation of a labeled nucleotide during amplification reaction is preferred because it increases the sensitivity of the assay with respect to extension labeling primers.

To overcome differences in DNA or RNA extraction and amplification efficiencies between independent samples, results can be normalized based on a suitable housekeeping gene control.

To enhance the reproducibility of the method, quality control should be performed by including a minimum of two negative controls and two positive controls in each assay.

PCR-CLEIA provides semiquantitative information on the target sequence. However, more accurate quantitative information can be obtained by adding an internal standard consisting of a competitor sequence of target DNA, which can be internally mutagenized. The coamplification of target and internal standard competitor sequences, using the same set of primers and the same physical-chemical parameters, allows the same efficiency of amplification and permits the construction of competitive titration curves for the quantitation of the target DNA.

Limit of Detection

Limits of detection of the PCR-CLEIA methods have to be determined using different concentrations of target nucleic acids. The products of amplification, once hybridized with the immobilized specific probes, are analyzed and the limit of detection for each amplicon is determined as the chemiluminescent signal significantly above cutoff. The criteria for defining the cutoff value and the positivity of samples have to be determined for each protocol based on the expected variability of the results.

Specificity

Serial dilutions of samples containing known numbers of copies of target genomes and negative controls (unrelated genomes) have to be PCR-amplified and hybridized with

CL substrate Light

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Getting Started With Dumbbells

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