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PCR Steps


Molecular Diagnostics and Genotyping

CHD is happy to add an aspect of molecular diagnostics to its repertoire of services.  Following published manuscripts, Cryptosporidium spp.1,2 and Giardia lamblia3 can be detected via the polymerase chain reaction (PCR) in treated, raw, and waste waters. In addition, speciation, lineage, and/or genotype determination are accomplished using restriction fragment length polymorphism (RFLP) and DNA sequencing. 


Figure: 1.5% EtBr stained agarose gel depicting Cryptosporidium species determination via RFLP digestion of a PCR amplified segment within the 18S rRNA gene.  

          

          Lane L: 50Bp Marker
          Lane 1: C. hominis PCR amplified product size: 593bp
          Lane 2: C. parvum PCR amplified product size 590bp
          Lane 3: C. baileyi PCR amplified product size 579bp
          Lane 4: C. serpentis PCR amplified product size 583bp
          B: Blank
          Lane 5: C. hominis amplified product digested with VspI enzyme
          Lane 6: C. parvum amplified product digested with VspI enzyme
          Lane 7: C. baileyi amplified product digested with VspI enzyme
          Lane 8: C. serpentis amplified product digested with VspI enzyme
          B: Blank
          Lane 9: C. hominis amplified product digested with DraII enzyme
          Lane 10: C. parvum amplified product digested with DraII enzyme
          Lane 11: C. baileyi amplified product digested with DraII enzyme
          Lane 12: C. serpentis amplified product digested with DraII enzyme


Figure 2: 0.8% EtBr stained agarose gel depicting an approximate 500bp size polymorphism within the GP900 surface protein gene noted between C. parvum and C. hominis.

                    Lane L: Marker, Lambda DNA digested with HindIII
                    Lane 1: C. parvum, Iowa Isolate (a.k.a. genotype II); Band Size: ~ 5100bp
                    Lane 2: C. hominis, Peruvian Isolate 278 (a.k.a. genotype I)
                    Lane 3: C. hominis, Peruvian Isolate 339 (a.k.a. genotype I)
                    Lane 4: C. hominis, Peruvian Isolate JOJ (a.k.a. genotype I)
                    Lane 5: C. hominis, Peruvian Isolate (a.k.a. genotype I)
                    Lane 6: C. hominis, Peruvian Isolate (a.k.a. genotype I)

 Peruvian isolates donated by Dr. Robert Gilman, Johns Hopkins University
 USA isolates donated by Dr. Michael Arrowood, C.D.C

 

1 Sturbaum, GD, C Reed, PJ Hoover, BH Jost, MM Marshall, and CR Sterling. 2001. Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts.  Applied and Environmental Microbiology. 67:2665-2668.

2 Gobet P, Toze S. Sensitive genotyping of Cryptosporidium parvum by PCR-RFLP analysis of the 70-kilodalton heat shock protein (HSP70) gene. FEMS Microbiol Lett 2001;200:37-41.

3 Hopkins RM, Meloni BP, Groth DM, Wetherall JD, Reynoldson JA, Thompson RC.
Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality.
Journal of Parasitology. 1997 Feb;83(1):44-51.

4 Lu SQ, Baruch AC, Adam RD. Molecular comparison of Giardia lamblia isolates. International Journal for Parasitology. 1998 Sep;28(9):1341-5.

 

 

Send mail to gsturbaum@chdiagnostic.com with questions or comments about this web site.
Copyright 2005 CH Diagnostic & Consulting Service, Inc.
Last modified: June 11, 2007