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PCR-ELISA - A low technology alternative to real time PCR
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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 email@example.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.
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