Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors - PubMed (original) (raw)

Comparative Study

Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors

Jianlin Jiang et al. Appl Environ Microbiol. 2005 Mar.

Abstract

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/mul or 25 ng of T4 gene 32 protein/mul to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.

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Figures

FIG. 1.

Impact of PCR inhibitors on PCR amplification. One (top) or two (bottom) microliters of 100 μl of DNA extracted with methods 4 (lanes 1 to 5), 3 (lanes 6 to 10), 2 (lanes 11 to 15), 6 (lanes 16 to 20), 5 (lanes 21 to 25), and 1 (lanes 26 to 30) from storm water samples seeded with 50 C. parvum oocysts was used for PCR analysis of the SSU rRNA gene without secondary purification of the DNA or the inclusion of facilitators in the PCR mixture. Lane 32 (top) was the positive PCR control (DNA of C. serpentis), and lanes 31 to 33 (bottom) were negative PCR controls.

FIG. 2.

Relief of PCR inhibitors with the inclusion of 400 (top) or 600 ng (bottom) of nonacetylated BSA/μl in the PCR mixture. Two microliters of 100 μl of DNA extracted with methods 4 (lanes 1 to 5), 3 (lanes 6 to 10), 2 (lanes 11 to 15), 6 (lanes 16 to 20), 5 (lanes 21 to 25), and 1 (lanes 26 to 30) from storm water samples seeded with 50 C. parvum oocysts was used for PCR analysis of the SSU rRNA gene. Lane 32 was a positive PCR control (DNA of C. serpentis), lane 31 was blank, and lanes 33 to 35 were negative PCR controls.

FIG. 3.

The presence of residual PCR inhibitors in DNA extracted by five DNA extraction methods. Two microliters of 100 μl of DNA extracted from a 0.5-ml pellet of _Cryptosporidium-_negative storm water samples by methods 1 (lanes 1 to 5), 5 (lanes 6 to 10), 3 (lanes 11 to 15), 4 (lanes 16 to 20), and 2 (lanes 21 to 25) was used for PCR amplification of the SSU rRNA gene. One microliter of DNA equivalent to five purified C. parvum oocysts was used as the PCR template without (top) or with (bottom) the inclusion of 400 ng of nonacetylated BSA/μl in the PCR mixture. Lane 27 was a positive PCR control (DNA equivalent to five purified C. parvum oocysts), lane 26 was blank, and lanes 28 to 30 were negative PCR controls.

FIG. 4.

Sensitivity of Cryptosporidium PCR detection with DNA extracted by methods 1 and 5. DNA was extracted from a _Cryptosporidium-_negative surface water sample seeded with 5 (lanes 1 to 5), 10 (lanes 6 to 10), 25 (lanes 11 to 15), and 50 (lanes 16 to 20) oocysts of C. parvum by DNA extraction methods 5 (top) and 1 (bottom). PCR analysis of the SSU rRNA gene was performed with the inclusion of 400 ng of nonacetylated BSA/μl. Lane 21 was a positive PCR control (DNA of C. serpentis), and lane 22 was one of the negative PCR controls.

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Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors - PubMed (original) (raw)