PCR-ELISA, an alternative to real time PCR for detecting products of the polymerase chain reaction


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	PCR-ELISA - A low technology alternative to real time PCR	BT&C Inc. 291 Route 22 East Building 6 Lebanon, NJ 08833 TEL: (908) 253-3444 FAX: (908) 575-1660 Email:info@btc-bti.com
HOME About BT&C Management Contact Us SERVICES Contract Research Consulting Product Development Custom Training CASE STUDIES Cytophobic Cultureware Keratinocyte Harvesting Defective Toy Absent Scientists LITERATURE Homogenizer Comparison Low Binding Beads Cell Based Assays High Throughput Grinding DNA Isolation from Soybeans Aseptic Freeze Drying Cryogenic Storage Homogenization Guide Maintenance of Protein Stability OPS Diagnostics Custom Products New Products Online Store Catalog	PCR-ELISAs (also known as PCR ELOSA) are a capture assay for nucleic acids that mimic enzyme linked immunosorbant assays. In this assay, PCR products hybridized to an immobilized capture probe. The assay thus measures sequences internal to the PCR product and is a less expensive assay and an alternative to real time PCR. PCR-ELISAs have been in use since the late 1980s and have developed into an assay for detecting specific sequences within polymerase chain reaction products. Though many methods are available for detecting specific sequences, PCR ELISAs are useful for detecting and differentiating between multiple targets. The PCR ELISA is also useful for screening multiple samples, especially when the number of samples does not warrant the expense of HTS methodologies (see reference). One of the most useful aspects of the PCR-ELISA is differentiating between polymerase chain reaction products generated from a common set of primers that contain sequence variations, i.e., sequence variation between the primers. As the assay plates are relatively inexpensive to prepare, capture probes can be added, deleted, and modified readily. Other recent work that has furthered PCR detection resulted in OPSD's low binding grinding beads. In 2002 we were asked to develop a product that could be used to shear mammalian DNA in order to increase the efficiency of a real time PCR process. This lead to our zirconium grinding beads. During subsequent research we found that by treating beads with a proprietary process, nonspecific binding of proteins and DNA could be reduced. Unexpectedly, these low binding grinding beads lowered the detection limit for cells that were homogenized by bead beating prior to real time PCR analysis. Apparently DNA and RNA liberated during the lysis of dilute concentration of cells was adsorbing to the grinding media, thus becoming unavailable for amplification. The treatment of the beads with a low binding material helped to remedy this problem. The following paper was published in Molecular and Cellular Probes, April 2000, Volume 14(2): 101-108. If you have any questions, please feel free to contact us at info@btc-bti.com. Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA). Zhang P, Gebhart CJ, Burden D, Duhamel GE. Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln 68583-0905, USA. Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide. Routine laboratory diagnosis of PE is done by amplification of L. intracellularis -specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide. We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L. intracellularis 328-bp PCR amplified products. The ELOSA involved determination of optical density value at 450 nm (OD(450)) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex. A positive ELOSA cut-off value of > or =0.375 was established using the mean OD(450)of negative control specimens plus three times the standard deviation. Using this value, the detection limit of PCR amplified L. intracellularis -specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6.1 ng, between 0.8 and 3.0 ng, and 0.8 ng of DNA, respectively. Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L. intracellularis -specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy. The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective interpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis. The following are several PCR ELISA publications obtained from Medline that illustrate the technology: 1. Hatano H, Kawashima H, Ogose A, Hotta T, Endo N. 2001. A PCR-ELISA assay for the detection of disseminated osteosarcoma cells in a mouse metastatic model. J Orthop Sci 6(3):269-275 2. Abe T, Yoshikawa T, Ihira M, Suzuki K, Suga S, Nishida M, Nagata M, Asano Y. 2001. Quantitation of human herpesvirus 6 DNA in infant with exanthem subitum by microplate PCR-hybridization assay. Pediatr Int Aug;43(4):372-378 3. Venturoli S, Cricca M, Bonvicini F, Gallinella G, Gentilomi G, Zerbini M, Musiani M. 2001. Detection of adeno-associated virus DNA in female genital samples by PCR-ELISA. J Med Virol Aug;64(4):577-582 4. Martin-Sanchez J, Lopez-Lopez MC, Acedo-Sanchez C, Castro-Fajardo JJ, Pineda JA, Morillas-Marquez F. 2001. Diagnosis of infections with Leishmania infantum using PCR-ELISA. Parasitology Jun;122( Pt 6):607-615 5. Laoboonchai A, Kawamoto F, Thanoosingha N, Kojima S, Scott Miller RR, Kain KC, Wongsrichanalai C. 2001. PCR-based ELISA technique for malaria diagnosis of specimens from Thailand. Trop Med Int Health Jun;6(6):458-462 6. Fach P, Perelle S, Dilasser F, Grout J. 2001. Comparison between a PCR-ELISA test and the vero cell assay for detecting Shiga toxin-producing Escherichia coli in dairy products and characterization of virulence traits of the isolated strains. J Appl Microbiol May;90(5):809-818 7. Zerbini M, Venturoli S, Cricca M, Gallinella G, De Simone P, Costa S, Santini D, Musiani M. 2001. Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA. J Clin Pathol May;54(5):377-380 8. Garcia L, Alonso-Sanz M, Rebollo MJ, Tercero JC, Chaves F. 2001. Mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates in Spain and their rapid detection by PCR-enzyme-linked immunosorbent assay. J Clin Microbiol May;39(5):1813-1818 9. Munch M, Nielsen LP, Handberg KJ, Jorgensen PH. 2001. Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA. Arch Virol 146(1):87-97 10. Shamloul AM, Abdallah NA, Madkour MA, Hadidi A. 2001. Sensitive detection of the Egyptian species of sugarcane streak virus by PCR-probe capture hybridization (PCR-ELISA) and its complete nucleotide sequence. J Virol Methods Mar;92(1):45-54	Products for PCR Low binding grinding beads for better protein and DNA yields Microplate Stability Chamber Reduce CVs and eliminate the Edge Effect from 96 well plates HT Homogenizer High throughput homogenization in microplate format

PCR-ELISA - A low technology alternative to real time PCR