Chapter 16 : Microwell Plate Detection Systems for Amplicon Detection and Characterization

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Microwell Plate Detection Systems for Amplicon Detection and Characterization, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap16-1.gif /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap16-2.gif


Sequence-specific methods for the detection of the products of nucleic acid amplification procedures (amplicons) have been developed for a variety of solid phases, including nylon membranes, microwell plates, microparticles, and, most recently, microchips (oligonucleotide probe microarrays). This chapter deals with procedures in which the molecules that capture amplicons are immobilized onto the surface of wells of microwell plates. Microwell plate detection systems can be divided into two formats based on the molecule used to capture amplicons: an oligonucleotide probe (sequence-specific capture) and avidin (nonspecific capture). Microwell plate detection systems have been developed in-house and are available commercially. Microwell plate detection procedures are frequently based on capture and detection of biotinylated amplicons. This requires that the primer used in the PCR amplification to generate the amplicon strand complementary to the probe be tagged with biotin. The biotin substituent will allow the amplicon to be either captured or detected by avidin (or streptavidin), depending on the detection format. Polystyrene microwell plates are typically used for amplicon detection because of their high DNA binding capabilities. There is substantial literature demonstrating the excellent analytical performance and clinical utility of microwell plate detection systems for PCR amplicon detection. Microwell plate detection of amplicons is flexible, is compatible with virtually any target, and can easily detect multiple pathogens under common hybridization and wash conditions.

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16

Key Concept Ranking

Human parainfluenza virus 1
Hepatitis C virus
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of FIGURE 1

Schematic diagrams of microwell plate detection using sandwich (A) and direct (B and C) hybridization formats.

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Bass, C. A.,, D. L. Jungkind,, N. S. Silverman, and, J. M. Bondi. 1993. Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens. J. Clin. Microbiol. 31: 26482653.
2. Bastien, P.,, G. W. Procop, and, U. Reischl. 2008. Quantitative real-time PCR is not more sensitive than “conventional” PCR. J. Clin. Microbiol. 46: 18971900.
3. Bobo, L.,, B. Munoz,, R. Viscidi,, T. Quinn,, H. Mkocha, and, S. West. 1991. Diagnosis of Chlamydia trachomatis eye infection in Tanzania by polymerase chain reaction/enzyme immunoassay. Lancet 338: 847850.
4. Buck, G. E. 1996. Detection of Bordetella pertussis by rapid-cycle PCR and colorimetric microwell hybridization. J. Clin. Microbiol. 34: 13551358.
5. Caliendo, A. M. 1995. Laboratory methods for quantitating HIV RNA. AIDS Clin. Care 7: 8991.
6. Chaharaein, B.,, A. R Omar,, L. Aini,, K. Yusoff, and, S. S. Hassan. 2009. Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction. Microbiol. Res. 164: 146179.
7. Chee, M. S.,, R. Yang,, E. Hubbell,, A. Berno,, X. C. Huang,, D. Stern,, J. Winkler,, D. J. Lockhart,, M. S. Morris, and, S. P. A. Fodor. 1996. Accessing genetic information with high-density DNA arrays. Science 274: 610614.
8. Chia, J. K.,, and A. Y. Chia. 2008. Chronic fatigue syndrome is associated with chronic enterovirus infection of the stomach. J. Clin. Pathol. 61: 4348.
9. Clinical and Laboratory Standards Institute. 2006. Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline2nd ed. CLSI document MM3-A2. Clinical and Laboratory Standards Institute, Wayne, PA.
10. Cook, A. F.,, E. Vuocolo, and, C. L. Brakel. 1988. Synthesis and hybridization of a series of biotinylated oligonucleotides. Nucleic Acids Res. 16: 40774095.
11. DiDomenico, N.,, H. Link,, R. Knobel,, T. Caratsch,, W. Weschler,, Z. G. Loewy, and, M. Rosenstraus. 1996. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR. Clin. Chem. 42: 19151923.
12. Elder, B. L.,, S. A. Hansen,, J. A. Kellogg,, F. J. Marsik, and, R. J. Zabransky. 1997. Cumitech 31, Verification and Validation of Procedures in the Clinical Microbiology Laboratory. Coordinating editor, B. W. McCurdy. ASM Press, Washington, DC.
13. Fan, J.,, K. J. Henrickson, and, L. L. Savatski. 1998. Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex). Clin. Infect. Dis. 26: 13971402.
14. Freymuth, F.,, G. Eugene,, A. Vabret,, J. Petitjean,, E. Gennetay,, J. Brouard,, J. F. Duhamel, and, B. Guillois. 1995. Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay. J. Clin. Microbiol. 33: 33523355.
15. Gerner, J. J.,, T. M. Gerads,, J. N. Mandrekar,, P. S. Mitchell, and, J. D. C. Yao. 2006. Detection of HIV-1 proviral DNA with the AMPLICOR HIV-1 DNA test, version 1.5, following sample processing by the MagNA Pure LC instrument. J. Clin. Virol. 37: 195198.
16. Hymas, W. C.,, W. K. Aldous,, E. W. Taggart,, J. B. Stevenson, and, D. R. Hillyard. 2008. Description and validation of a novel real-time RT-PCR enterovirus assay. Clin. Chem. 54: 406413.
17. Inouye, S.,, and R. Hondo. 1990. Microplate hybridization of amplified viral DNA segment. J. Clin. Microbiol. 28: 14691472.
18. Kawai, S.,, S. Maekawajiri, and, A. Yamane. 1993. A simple method of detecting amplified DNA with immobilized probes on microtiter wells. Anal. Biochem. 209: 6369.
19. Keller, G. H.,, D. P. Huang, and, M. M. Manak. 1989. A sensitive nonisotopic hybridization assay for HIV-1 DNA. Anal. Biochem. 177: 2732.
20. Keller, G. H.,, D. P. Huang, and, M. M. Manak. 1991. Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells. J. Clin. Microbiol. 29: 638641.
21. Keller, G. H.,, D. P. Huang,, J. W. K. Shih, and, M. M. Manak. 1990. Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization. J. Clin. Microbiol. 28: 14111416.
22. Khanna, M.,, J. Fan,, K. Pehler-Harrington,, C. Waters,, P. Douglass,, J. Stallock,, S. Kehl, and, K. J. Henrickson. 2005. The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time counterpart. J. Clin. Microbiol. 43: 565571.
23. Kim, H. J.,, and J. C. Cho. 2008. Rapid and sensitive detection of Listeria monocytogenes using a PCR-enzyme linked immunosorbent assay. J. Microbiol. Biotechnol. 18: 18581861.
24. Landry, M. L.,, R. Garner, and, D. Ferguson. 2003. Comparison of the NucliSens basic kit (nucleic acid sequence-based amplification) and the Argene Biosoft enterovirus consensus reverse transcription-PCR assays for rapid detection of enterovirus RNA in clinical specimens. J. Clin. Microbiol. 41: 50065010.
25. Li, H. J.,, J. S. Dummer,, W. R. Estes,, S. F. Meng,, P. F. Wright, and, Y. W. Tang. 2003. Cytomegalovirus loads measured by a quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J. Clin. Microbiol. 41: 187191.
26. Liolios, L.,, A. Jenney,, D. Spelman,, T. Kosimbos,, M. Catton, and, S. Wesselingh. 2001. Comparison of a multiplex reverse transcription-PCR enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens. J. Clin. Microbiol. 39: 27792783.
27. Loeffelholz, M. J.,, C. J. Thompson,, K. S. Long, and, M. J. R. Gilchrist. 1999. Comparison of PCR, culture, and direct fluorescent-antibody testing for detection of Bordetella pertussis. J. Clin. Microbiol. 37: 28722876.
28. Milne, S. A.,, S. Gallacher,, P. Cash,, D. N. Lees,, K. Henshilwood, and, A. J. Porter. 2007. A sensitive and reliable reverse transcriptase PCR-enzyme-linked immunosorbent assay for the detection of human pathogenic viruses in bivalve molluscs. J. Food Prot. 70: 14751482.
29. Musiani, M.,, G. Gallinella,, S. Venturoli, and, M. Zerbini. 2007. Competitive PCR-ELISA protocols for the quantitative and the standardized detection of viral genomes. Nat. Protoc. 2: 25112519.
30. Nagata, Y.,, H. Yokota,, O. Kosuda,, K. Yokoo,, K. Takemura, and, T. Kikuchi. 1985. Quantification of picogram levels of specific DNA immobilized in microtiter wells. FEES Lett. 183: 379382.
31. Poehling, K. A.,, M. R. Griffin,, R. S. Dittus,, Y. W. Tang,, K. Holland,, H. J. Li, and, K. M. Edwards. 2002. Bedside diagnosis of influenza virus infections in hospitalized children. Pediatrics 110: 8388.
32. Rahimian, J.,, D. Raoult,, Y. W. Tang, and, B. A. Hanna. 2006. Bartonella quintana endocarditis with positive serology for Coxiella burnetii. J. Infect. 53: e151e153.
33. Rapier, J. M.,, Y. Villamarzo,, G. Schochetman,, C. Y. Ou,, C. L. Brakel,, J. Donegan,, W. Maltzman,, S. Lee,, D. Kirtikar, and, D. Gatica. 1993. Nonradioactive, colorimetric microplate hybridization assay for detecting amplified human immunodeficiency virus DNA. Clin. Chem. 39: 244247.
34. Santos, N.,, S. Honma,, C. Timenetsky Mdo,, A. C. Linhares,, H. Ushijima,, G. E. Armah,, J. R. Gentsch, and, Y. Hoshino. 2008. Development of a microtiter plate hybridization-based PCR-enzyme linked immunosorbent assay for identification of clinically relevant human group A rotavirus G and P genotypes. J. Clin. Microbiol. 46: 462469.
35. Schachter, J.,, W. E. Stamm,, T. C. Quinn,, W. W. Andrews,, J. D. Burczak, and, H. H. Lee. 1994. Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix. J. Clin. Microbiol. 32: 25402543.
36. Schutzle, H.,, J. Weigl,, W. Puppe,, J. Forster, and, R. Berner. 2008. Diagnostic performance of a rapid antigen test for RSV in comparison with a 19-valent multiplex RT-PCR ELISA in children with acute respiratory tract infections. Eur. J. Pediatr. 167: 745749.
37. Stellrecht, K. A.,, I. Harding,, F. M. Hussain,, N. G. Mishrik,, R. T. Czap,, M. L. Lepow, and, R. A. Venezia. 2000. A one-step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis. J. Clin. Virol. 17: 143149.
38. Stellrecht, K. A.,, A. M. Woron,, N. G. Mishrik, and, R. A. Venezia. 2004. Comparison of multiplex PCR assay with culture for detection of genital mycoplasmas. J. Clin. Microbiol. 42: 15281533.
39. St-Louis, M. 2009. PCR-ELISA for high-throughput blood group genotyping. Methods Mol. Biol. 496: 313.
40. Tang, Y. W.,, P. J. Heimgartner,, S. J. Tollefson,, T. J. Berg,, P. N. Rys,, H. J. Li,, T. F. Smith,, D. H. Persing, and, P. F. Wright. 1999. A colorimetric microtiter plate PCR system detects respiratory syncytial virus in nasal wash specimens and discriminates subtypes A and B. Diagn. Microbiol. Infect. Dis. 34: 333337.
41. Tang, Y. W.,, J. E. Johnson,, P. J. Browning,, R. Cruz-Gervis,, A. Davis,, B. S. Graham,, K. L. Brigham,, J. A. Oates,, J. E. Loy, and, A. A. Stecenko. 2003. Herpesvirus DNA is consistently detected in lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Microbiol. 41: 26332640.
42. Tang, Y. W.,, H. J. Li,, M. M. Durkin,, S E. Sefers,, S. Meng,, P. A. Connolly,, C. W. Stratton, and, L. J. Wheat. 2006. Detection of Histoplasma capsulatum in urine by PCR and its role in the diagnosis of histoplasmosis. Diagn. Microbiol. Infect. Dis. 54: 283287.
43. Tang, Y. W.,, H. J. Li,, A. Roberto,, D. Warner, and, B. Yen-Lieberman. 2004. Detection of hepatitis C virus by a user-developed reverse transcriptase-PCR and use of amplification products for subsequent genotyping. J. Clin. Virol. 31: 148152.
44. Tang, Y. W.,, S. Meng,, H. J. Li,, C. W. Stratton,, T. Koyamatsu, and, X. Zheng. 2004. PCR enhances acid-fast bacillus stain-based rapid detection of Mycobacterium tuberculosis. J. Clin. Microbiol. 42: 18491850.
45. Tang, Y. W.,, P. N. Rys,, B. J. Rutledge,, P. S. Mitchell,, T. F. Smith, and, D. H. Persing. 1998. Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid. J. Clin. Microbiol. 36: 27142717.
46. Tsang, R. S.,, C. M. Tsai,, A. M. Henderson,, S. Tyler,, D. K. Law,, W. Zolliinger, and, F. Jamieson. 2008. Immunochemical studies and genetic background of two Neisseria meningitidis isolates expressing unusual capsule polysaccharide antigens with specificities of both serogroup Y and W135. Can. J. Microbiol. 54: 229234.
47. Wang, E.,, S. Paessler,, P. Aguilar,, A. S. Carrara,, H. Ni,, I. P. Greene, and, S. C. Weaver. 2006. Reverse transcription-PCR-enzyme-linked immunosorbent assay for rapid detection and differentiation of alphavirus infections. J. Clin. Microbiol. 44: 40004008.
48. White, T. J.,, R. Madej, and, D. H. Persing. 1992. The polymerase chain reaction: clinical applications. Adv. Clin. Chem. 29: 161196.
49. Wilson, T.,, and J. Carson. 2003. Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridization assay to detect selected bacterial fish pathogens. Dis. Aquat. Organ. 54: 127134.


Generic image for table

Conditions for immobilization of DNA probes in microwell plates

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16
Generic image for table

Microwell plate hybridization conditions

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16
Generic image for table

Advantages and disadvantages of microwell plate detection systems versus alternatives

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error