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target-specific probes. A positive signal has to be detected when target genome PCR amplicons are hybridized with the specific probes. A signal within background range (negative signal) has to be detected when PCR amplicons from unrelated genomes are set for hybridization with target-specific probes.

Precision

To investigate the reproducibility of the PCR-CLEIA method, at least two positive and two negative reference samples have to be amplified, and then assayed with type-specific immobilized oligoprobes in triplicate in three independent assays. Intraassay coefficient of variation (CV) lower than 5% and interassay CV lower than 10% are desirable.

Applications of PCR-CLEIA

PCR-CLEIA has been developed and applied to the detection of PCR-amplified enterotoxin A gene from Clostridium perfringens in artificially contaminated ground beef.[3] A biotinylated primer pair was designed for the amplification of a fragment of the C. perfringens enterotoxin A gene. PCR-amplified products were detected by hybridization ELISA protocols by applying a streptavidin capture step for the hybridized PCR products to an internal Dig-labeled probe using chemiluminescent detection with Lumiphos 530TM as substrate.

Quantification of circulating human cytomegalovirus (HCMV) in the plasma of severely leukopenic patients was also achieved by PCR-CLEIA through the detection of biotinylated PCR products of the HCMV UL50 region using HCMV-specific enzyme-labeled probes and automated chemiluminescence detection able to reveal 100 HCMV genomes per milliliter of plasma.[4]

A sensitive PCR-CLEIA assay was developed and applied to quantitative detection of enteroviruses in environmental samples.[5] Following reverse transcription (RT), viral cDNA was labeled with Dig-dUTP during the PCR amplification step. The labeled PCR products were then hybridized with enterovirus-specific biotinylated probe, captured in streptavidin-coated microtiter wells, and detected by an anti-Dig peroxidase conjugate using a chemiluminescent substrate and automated measurement. The assay was able to detect 0.01 plaque-forming units (PFU)/mL, proving 10-100 times more sensitive than colorimetric detection or dot blot hybridization.

PCR-CLEIA has proved to be a sensitive and versatile method for the analysis of human and murine cytokine mRNA expression. Indeed, Dufour et al.[6] were able to quantify mRNA for five porcine cytokines: interferon

(IFN)-gamma, interleukin (IL)-2, IL-4, IL-10, and IL-18 on peripheral blood mononuclear cells. The main features of the methodology were: RT to obtain DNA sequences, PCR and detection of amplicons for all cytokines simultaneously, cytokine quantification in relation to a housekeeping gene control (glyceraldehyde-3-phosphate dehydrogenase, or GAPDH), detection of amplicons by ELISA using a chemiluminescent substrate with high sensitivity and wide dynamic range, and automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative RT-PCR-CLEIA was able to detect 100-200 cytokines mRNA copies per 7.5 x 104 cells.

Immunochemiluminescent PCR detection of varicella zoster virus (VZV) DNA from the cerebrospinal fluid of 287 patients with meningitis, encephalitis, or other neurological diseases or symptoms improved considerably the detection rate of the VZV-PCR product compared with agarose gel electrophoresis.[7]

Hepatitis G virus (HGV) was recently identified as a new member of the family Flaviviridae, but its clinical significance is still unclear. Because no immunoassay for the diagnosis of HGV is available, to facilitate the detection of the viral genome by mass screening in the clinical laboratory, a sensitive RT-PCR assay with ELISA detection was developed. Sequences within the 5'-non-coding region and within the putative NS5a region were independently amplified in the presence of Dig-dUTP and were detected by hybridization with biotinylated capture probes bound to a streptavidin-coated matrix. Semiquantitative detection via chemiluminescence was performed in microtiter plate format machines. At least 8 x 102 genome equivalents per milliliter of serum using both primer pairs could be detected.[8]

The PCR-CLEIA methods described above were developed using the conventional 96-well microtiter plate format. However, other assay formats can be explored, thanks to the use of chemiluminescence detection, thus enhancing the performance of the method.

For example, using multianalyte assay formats and type-specific probes, PCR-CLEIA can also be easily applied to the simultaneous identification of the different genotypes of pathogens and can therefore be a valid tool to study the prevalence and potential pathogenicity of single genotypes. This is possible, thanks to the use of chemiluminescence as detection system, which allows both quantification and localization of the hybrids captured onto the plate.[9] In particular, to allow multi-analyte binding assays, we have developed a novel microtiter plate containing 24 main wells, each divided into seven subwells, and we explored its clinical potential by developing a PCR-CLEIA for simultaneous detection and typing of seven high-oncogenic-risk human

Fig. 2 Multititer plate figure. Analysis of clinical samples [n = 24; 5 negative and 19 positive (five for HPV 16, three for HPV 18, two for HPV 31, three for HPV 33, one for HPV 35, two for HPV 45, one for HPV 58, one for HPV 16 and 18, and one for HPV 16 and 31)] performed on the novel microtiter plate format for multianalyte assays. Overlay image of the plate live image and the chemiluminescent signal. White letters indicate HPV genotypes detected in the subwells of each main well, based on the immobilized type-specific oligoprobe positions: (a) HPV 16; (b) HPV 18; (c) HPV 31; (d) HPV 33; (e) HPV 35; (f) HPV 45; (g) HPV 58.

Fig. 2 Multititer plate figure. Analysis of clinical samples [n = 24; 5 negative and 19 positive (five for HPV 16, three for HPV 18, two for HPV 31, three for HPV 33, one for HPV 35, two for HPV 45, one for HPV 58, one for HPV 16 and 18, and one for HPV 16 and 31)] performed on the novel microtiter plate format for multianalyte assays. Overlay image of the plate live image and the chemiluminescent signal. White letters indicate HPV genotypes detected in the subwells of each main well, based on the immobilized type-specific oligoprobe positions: (a) HPV 16; (b) HPV 18; (c) HPV 31; (d) HPV 33; (e) HPV 35; (f) HPV 45; (g) HPV 58.

papillomavirus (HPV) DNAs in one well. The assay is based on consensus PCR amplification of a conserved sequence of 30 HPV genital genotypes and on the typing of seven HPV DNA using the novel microtiter plate format. Thanks to the possibility of immobilizing seven type-specific capture oligoprobes in separate positions within the same well, typing was performed simultaneously by hybridization followed by a chemiluminescent immunoassay in one well and with one aliquot of the amplification reaction product. The chemiluminescence signal was imaged using an ultrasensitive charge-coupled device (CCD) camera and the light output intensity of each internal subwell was measured. The method proved to be specific and allowed the detection of 50 copies of genome for HPV 16, 18, 33, and 58, and 100 copies of genome for HPV 31, 35, and 45. Intraassay and interassay coefficients of variation of the method were 5.6% and 7.9%, respectively. This new assay format offers advantages in terms of multitest simplification and reductions in reagent volumes and analysis time. The same principle could be applied to the development of single-tube panels of tests for other pathological conditions.

Figure 2 shows the results obtained by analyzing 24 clinical samples with the multianalyte PCR-CLEIA assay, developed using the novel microtiter plate format. In particular, the overlay image of the plate live image and the chemiluminescent signal is shown.

Future trends for PCR-CLEIA mainly involve the development of miniaturized and high-throughput assay formats. With respect to this, we are now developing a PCR-CLEIA on 384-well microtiter plates, which allows to greatly increase the amount of information obtained in one assay. The use of smaller assay volumes allowed us to significantly reduce the amount of samples and reagents, thus providing savings. This format allows the screening of 20 samples for as many as 15 HPV genotypes per sample in one assay and with one 50-p.L amplification product aliquot. Moreover, thanks to the high density of the format, it is possible to produce calibration curves for each genotype and to obtain semiquantitative information on the viral load of positive samples. It is worth noting that this assay format provides optimal performance only when chemiluminescence is used as a detection system, thanks to its high detectability even in low volumes.

The use of robotic samples and reagent handling systems will enhance the throughput of the assay and allow the operator to control the analytical steps and the reproducibility of the overall procedure.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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