Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water

Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.

[1]  J. Rose,et al.  Evaluation of immunofluorescence techniques for detection of Cryptosporidium oocysts and Giardia cysts from environmental samples , 1989, Applied and environmental microbiology.

[2]  R M Atlas,et al.  Differentiation of Giardia duodenalis from other Giardia spp. by using polymerase chain reaction and gene probes , 1992, Journal of clinical microbiology.

[3]  W. Vahjen,et al.  Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast , 1993, Applied and environmental microbiology.

[4]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[5]  M. Woodward,et al.  Detection of Cryptosporidium parvum using a specific polymerase chain reaction. , 1993, Veterinary parasitology.

[6]  K. Botzenhart,et al.  Selective detection of viable Cryptosporidium oocysts by PCR. , 1994, Zentralblatt fur Hygiene und Umweltmedizin = International journal of hygiene and environmental medicine.

[7]  Jt. Lisle,et al.  Cryptosporidium contamination of water in the USA and UK : A mini-review , 1995 .

[8]  J. Rose,et al.  Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples , 1995, Applied and environmental microbiology.

[9]  N. Ashbolt,et al.  Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. , 1994, Cytometry.

[10]  R M Atlas,et al.  Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts , 1991, Applied and environmental microbiology.

[11]  B. K. Pal,et al.  The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. , 1991, DNA and cell biology.

[12]  C. Kreader.  Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein , 1996, Applied and environmental microbiology.

[13]  W. Liesack,et al.  Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment , 1992, Journal of bacteriology.

[14]  Barbara L. Herwaldt,et al.  Waterborne disease in the United States, 1991 and 1992 , 1994 .

[15]  J. Rose,et al.  DNA Probe Hybridization and PCR Detection of Cryptosporidium Compared to Immunofluorescence Assay , 1993 .

[16]  William D. Gollnitz,et al.  COMMERCIAL LABS: HOW ACCURATE ARE THEY? , 1994 .

[17]  B. Timblin,et al.  DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction. , 1991, The American journal of tropical medicine and hygiene.

[18]  M. Braun,et al.  Increasing proportions of uracil in DNA substrates increases inhibition of restriction enzyme digests. , 1994, BioTechniques.

[19]  D. Persing,et al.  Diagnostic molecular microbiology : principles and applications , 1993 .

[20]  D. Warhurst,et al.  Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction , 1994, Parasitology.

[21]  B H Olson,et al.  Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction , 1992, Applied and environmental microbiology.

[22]  G. Jenkins,et al.  Extraction and Amplification of 16S rRNA Genes from Deep Marine Sediments and Seawater to Assess Bacterial Community Diversity , 1995 .

[23]  X. Leng,et al.  Simplified method for recovery and PCR detection of Cryptosporidium DNA from bovine feces , 1996, Applied and environmental microbiology.

[24]  T. Nash,et al.  Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction. , 1992, Molecular and biochemical parasitology (Print).

[25]  Comparison of Two Methods for Detection ofGiardiaCysts andCryptosporidiumOocysts in Water , 1995 .

[26]  Ross A. Overbeek,et al.  The ribosomal database project , 1992, Nucleic Acids Res..

[27]  E. Carrington,et al.  Comparison of Methods for Recovery of Cryptosporidium from Water , 1993 .

[28]  C. M. Collins.  Waterborne disease. , 1985, The New Zealand medical journal.

[29]  E. C. Nieminski,et al.  Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water , 1995, Applied and environmental microbiology.