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A salting out and resin procedure for extracting Schistosoma mansoni DNA from human urine samples.

Enk MJ, Oliveira E Silva G, Rodrigues NB - BMC Res Notes (2010)

Bottom Line: The description of the extraction procedure is given.This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples.Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Esquistossomose - Centro de Pesquisas René Rachou (CPqRR) - Fundação Oswaldo Cruz (FIOCRUZ), Av, Augusto de Lima 1715, Belo Horizonte, Minas Gerais, 30190-002, Brazil. marenk@cpqrr.fiocruz.br.

ABSTRACT

Background: In this paper a simple and cheap salting out and resin (InstaGene matrix(R) resin - BioRad) DNA extraction method from urine for PCR assays is introduced. The DNA of the fluke Schistosoma mansoni was chosen as the target since schistosomiasis lacks a suitable diagnostic tool which is sensitive enough to detect low worm burden. It is well known that the PCR technique provides high sensitivity and specificity in detecting parasite DNA. Therefore it is of paramount importance to take advantage of its excellent performance by providing a simple to handle and reliable DNA extraction procedure, which permits the diagnosis of the disease in easily obtainable urine samples.

Findings: The description of the extraction procedure is given. This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples. The reproducibility reached 100%, showing positive results in 5 assay repetitions of 5 tested samples each containing 20 ng DNA/5 ml. The efficiency of the extraction procedure was also evaluated in a serial dilution of the original 20 ng DNA/5 ml sample. Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure.

Conclusions: This methodology represents a promising tool for schistosomiasis diagnosis utilizing a bio-molecular technique in urine samples which is now ready to be tested under field conditions and may be applicable to the diagnosis of other parasitic diseases.

No MeSH data available.


Related in: MedlinePlus

An 8% polyacrylamide gel, representative of 5 assays, showing the efficiency test of the extraction method. L = 100 bp Ladder, 1-6 = 2nd sample set, 4 ng/mL, 800 pg/mL, 160 pg/mL, 32 pg/mL, 6,4 pg/mL and 1.28 pg/mL of DNA respectively. - = negative control. + = S. mansoni DNA positive control.
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Figure 2: An 8% polyacrylamide gel, representative of 5 assays, showing the efficiency test of the extraction method. L = 100 bp Ladder, 1-6 = 2nd sample set, 4 ng/mL, 800 pg/mL, 160 pg/mL, 32 pg/mL, 6,4 pg/mL and 1.28 pg/mL of DNA respectively. - = negative control. + = S. mansoni DNA positive control.

Mentions: Positive results were observed for all 5 samples from the 1st set (figure 1), as well as for all 6 samples from 2nd set (figure 2) in all of the 5 assay repetitions. The results from the 1st sample set show the high reproducibility of the DNA extraction method, and those from the 2nd confirm the test's efficiency by detecting 1.28 pg DNA/mL urine, an approximately 3,000 times smaller quantity of DNA than in the first dilution (results samples set 2). Furthermore, these data confirm the PCR reproducibility and sensitivity, hence, parasite DNA was detected in all 5 assay repetitions.


A salting out and resin procedure for extracting Schistosoma mansoni DNA from human urine samples.

Enk MJ, Oliveira E Silva G, Rodrigues NB - BMC Res Notes (2010)

An 8% polyacrylamide gel, representative of 5 assays, showing the efficiency test of the extraction method. L = 100 bp Ladder, 1-6 = 2nd sample set, 4 ng/mL, 800 pg/mL, 160 pg/mL, 32 pg/mL, 6,4 pg/mL and 1.28 pg/mL of DNA respectively. - = negative control. + = S. mansoni DNA positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2877055&req=5

Figure 2: An 8% polyacrylamide gel, representative of 5 assays, showing the efficiency test of the extraction method. L = 100 bp Ladder, 1-6 = 2nd sample set, 4 ng/mL, 800 pg/mL, 160 pg/mL, 32 pg/mL, 6,4 pg/mL and 1.28 pg/mL of DNA respectively. - = negative control. + = S. mansoni DNA positive control.
Mentions: Positive results were observed for all 5 samples from the 1st set (figure 1), as well as for all 6 samples from 2nd set (figure 2) in all of the 5 assay repetitions. The results from the 1st sample set show the high reproducibility of the DNA extraction method, and those from the 2nd confirm the test's efficiency by detecting 1.28 pg DNA/mL urine, an approximately 3,000 times smaller quantity of DNA than in the first dilution (results samples set 2). Furthermore, these data confirm the PCR reproducibility and sensitivity, hence, parasite DNA was detected in all 5 assay repetitions.

Bottom Line: The description of the extraction procedure is given.This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples.Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Esquistossomose - Centro de Pesquisas René Rachou (CPqRR) - Fundação Oswaldo Cruz (FIOCRUZ), Av, Augusto de Lima 1715, Belo Horizonte, Minas Gerais, 30190-002, Brazil. marenk@cpqrr.fiocruz.br.

ABSTRACT

Background: In this paper a simple and cheap salting out and resin (InstaGene matrix(R) resin - BioRad) DNA extraction method from urine for PCR assays is introduced. The DNA of the fluke Schistosoma mansoni was chosen as the target since schistosomiasis lacks a suitable diagnostic tool which is sensitive enough to detect low worm burden. It is well known that the PCR technique provides high sensitivity and specificity in detecting parasite DNA. Therefore it is of paramount importance to take advantage of its excellent performance by providing a simple to handle and reliable DNA extraction procedure, which permits the diagnosis of the disease in easily obtainable urine samples.

Findings: The description of the extraction procedure is given. This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples. The reproducibility reached 100%, showing positive results in 5 assay repetitions of 5 tested samples each containing 20 ng DNA/5 ml. The efficiency of the extraction procedure was also evaluated in a serial dilution of the original 20 ng DNA/5 ml sample. Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure.

Conclusions: This methodology represents a promising tool for schistosomiasis diagnosis utilizing a bio-molecular technique in urine samples which is now ready to be tested under field conditions and may be applicable to the diagnosis of other parasitic diseases.

No MeSH data available.


Related in: MedlinePlus