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Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

ABSTRACT: BACKGROUND: The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis. FINDINGS: Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence. CONCLUSION: Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis. One needs to be aware of possible bacterial contamination when interpreting abnormal findings following PCR amplification of buccal swab DNA samples.

SUBMITTER: Mahfuz I

PROVIDER: S-EPMC4222080 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

Mahfuz Istiak I Cheng Wei W White Stefan J SJ

BMC research notes 20131122

<h4>Background</h4>The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis.<h4>Findings</h4>Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence.<h4>Conclusion</h4>C ...[more]

PMID: 24266944

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Project description:BackgroundIn 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections.ResultsDNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 ?g of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay.ConclusionsMouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.

Project description:Streptococcus parasanguinis is a dominant isolate of dental plaque and an opportunistic pathogen associated with subacute endocarditis. As the expression of collagen binding proteins (CBPs) could promote the establishment of S. parasanguinis in the host, the functions of three putative CBP-encoding loci, Spaf_0420, Spaf_1570, and Spaf_1573, were analyzed using isogenic mutant strains. It was revealed that S. parasanguinis FW213 bound effectively to fibronectin and type I collagen, but the strain's affinity for laminin and type IV collagen was quite low. By using various deletion derivatives, it was found that these three loci mediated the binding of S. parasanguinis to multiple extracellular matrix molecules, with type I collagen as the common substrate. Derivative strains with a deletion in any of the three loci expressed reduced binding to trypsin-treated swine heart valves. The deletion of these loci also reduced the viable count of S. parasanguinis bacteria within macrophages, especially the loss of Spaf_0420, but only strains with deletions in Spaf_0420 and Spaf_1570 expressed reduced virulence in the Galleria mellonella larva model. The deletion of Spaf_1570 and Spaf_1573 affected mainly the structure, but not the overall mass, of biofilm cultures in a flow cell system. Thus, CBPs are likely to be more critical for the initial colonization of S. parasanguinis on host tissues during the development of endocarditis.IMPORTANCE Bacteria generally can utilize multiple adhesins to establish themselves in the host. We found that Streptococcus parasanguinis, a dominant oral commensal and an opportunistic pathogen for subacute endocarditis, possesses at least three collagen-binding proteins that enable S. parasanguinis to successfully colonize damaged heart tissues and escape innate immune clearance. The binding specificities of these three proteins for extracellular matrix molecules differ, although all three proteins participate in biofilm formation by S. parasanguinis The "multiligand for multisubstrate" feature of these adhesins may explain the high adaptability of this microbe to different tissue sites.

Dataset Information

Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

Publications

Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

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