Comparison of DNA polymerases for improved forensic analysis of challenging samples

Nilsson, M.E.; Grånemo, Joakim; Buś, Magdalena M.; Havsjö, Mikael; Allen, Marie

doi:10.1016/j.fsigen.2016.05.017

Cited by 16 publications

(12 citation statements)

References 17 publications

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“…4,9,16,17,19, and 24) produced relatively low levels of nitrite (<0.5 mM Δ nitrite), while seven (Nos. 1,5,7,14,20,21, and 23) produced relatively high levels (>1.0 mM Δ nitrite) ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the reverse primer VR (5'-GTGTAACAAGGGAGTACGGACC-3') was used for all species (19). The expected molecular weights of the PCR products were 384 bp for V. atypica, 321 bp for V. dispar, and 604 bp for V. rogosae (20). The primers were synthesized by BEX Co. Ltd. (Tokyo, Japan).…”

Section: Direct Polymerase Chain Reaction (Pcr) Protocolmentioning

confidence: 99%

“…The PCR mixture (50 µL) was composed of 24 µL H 2 O, 12 µL bacterial slurry serially diluted 10-fold with PBS (10 −1 and 10 −2 ), 12 µL direct PCR kit (KAPA2G Robust HotStart ReadyMix with dye, Kapa Biosystems, Cape Town, South Africa), and 1 µL of each primer (50 pmol/µL). This PCR polymerase offers high amplification efficiency even in the presence of an inhibitor and is a useful tool for forensic samples (20).…”

Section: Direct Polymerase Chain Reaction (Pcr) Protocolmentioning

confidence: 99%

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Salivary nitrate-nitrite conversion capacity after nitrate ingestion and incidence of <i>Veillonella</i> spp. in elderly individuals

Mitsui

Saito

Harasawa³

2018

J Oral Sci

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Dietary nitrate has several beneficial effects, including blood pressure reduction and improved oxygen consumption efficiency, but in order to do so it must first be reduced to nitrite by oral bacteria. Veillonella spp., a strictly anaerobic group, are the most prevalent nitrate-reducing bacteria in the oral cavity. In response to some early studies that have hinted at inter- and intra-individual variation in salivary nitrate-nitrite conversion capacity, the purpose of the present study was to investigate the incidence of and variation in the Veillonella species V. atypica, V. dispar, and V. rogosae by direct PCR and to assess salivary nitrate-nitrite conversion capacity and its reproducibility after dietary nitrate consumption in 24 elderly individuals. V. atypica, V. dispar, and V. rogosae were detected in 10 (41.7%), 24 (100%), and 14 (58.3%) participants, respectively. The coefficients of correlation between the first and second experiments on increased nitrate/nitrite were 0.637 and 0.583, respectively, both of which were statistically significant (P < 0.01). In both experiments, 6 participants produced relatively low levels of nitrite (<0.5 mM Δ nitrite) while 7 produced relatively high levels (>1.0 mM Δ nitrite). The data suggested that V. dispar was the most prevalent species, being present even in individuals producing low levels of salivary nitrite.

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Section: Resultsmentioning

confidence: 99%

Section: Direct Polymerase Chain Reaction (Pcr) Protocolmentioning

confidence: 99%

Section: Direct Polymerase Chain Reaction (Pcr) Protocolmentioning

confidence: 99%

See 1 more Smart Citation

Salivary nitrate-nitrite conversion capacity after nitrate ingestion and incidence of <i>Veillonella</i> spp. in elderly individuals

Mitsui

Saito

Harasawa³

2018

J Oral Sci

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“…The KAPA3G Plant DNA polymerase is a high-efficiency polymerase formulated to improve tolerance to PCR inhibitors such as polyphenolics and polysaccharides and has previously permitted successful amplification with challenging samples [66,67]. Therefore, we assessed the performance of this polymerase on the untreated/unpurified old plant extract.…”

Section: Specialized Polymerasementioning

confidence: 99%

A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils

et al. 2018

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Soil is often collected from a suspect's tire, vehicle, or shoes during a criminal investigation and subsequently submitted to a forensic laboratory for analysis. Plant and insect material recovered in such samples is rarely analyzed, as morphological identification is difficult. In this study, DNA barcoding was used for taxonomic identifications by targeting the gene regions known to permit discrimination in plants [maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL)] and insects [cytochrome oxidase subunit I (COI)]. A DNA barcode protocol suitable for processing forensic-type biological fragments was developed and its utility broadly tested with forensic-type fragments (e.g., seeds, leaves, bark, head, legs; n, 213) isolated from soils collected within Virginia, USA (n, 11). Difficulties with PCR inhibitors in plant extracts and obtaining clean Sanger sequence data from insect amplicons were encountered during protocol development; however, the final protocol produced sequences specific to the expected locus and taxa. The overall quantity and quality of DNA extracted from the 213 forensic-type biological fragments was low (< 15 ng/μL). For plant fragments, only the rbcL sequence data was deemed reliable; thus, taxonomic identifications were limited to the family level. The majority of insect sequences matched COI in both GenBank and Barcode of Life DataSystems; however, they were identified as an undescribed environmental contaminant. Although limited taxonomic information was gleaned from the forensic-type fragments processed in this study, the new protocol shows promise for obtaining reliable and specific identifications through DNA barcoding, which could ultimately enhance the information gleaned from soil examinations.

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“…This PCR system has high amplification efficiency even in the presence of an inhibitor, and is a useful tool for forensic samples (15).…”

Section: Polymerase Chain Reaction (Pcr) Protocol and Semiquantificatmentioning

confidence: 99%

The effects of essential oil, povidone-iodine, and chlorhexidine mouthwash on salivary nitrate/nitrite and nitrate-reducing bacteria

Mitsui

Harasawa²

2017

J Oral Sci

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Dietary nitrate is reduced to nitrite and nitric oxide by microbial flora, and this activity is beneficial to vascular health. It has been reported that this bacterial process is inhibited by chlorhexidine mouthwash, although the effects of other products are largely unknown. This study examined the effects of several treatments on salivary nitrate/nitrite and nitrate-reducing bacteria. Twelve university staff and students performed mouth-washing with water (control), essential oil, 0.35% povidone-iodine, or 0.0025% chlorhexidine and then ate 100 g lettuce (110 mg nitrate content), followed by collection of saliva and tongue bacteria at the baseline, and 1, 5, and 10 h thereafter. The individual treatments were separated by an interval of one week. Salivary nitrate/nitrite was measured by the calorimetric method, and a representative nitrate-reducing bacterial species, Veillonella dispar, was detected and semi-quantified using a polymerase chain reaction (PCR) assay. Significant increases in salivary nitrate/nitrite were observed for all treatments (all P < 0.05). The PCR assay showed that water, essential oil, and povidone-iodine mouthwash had little effect, whereas V. dispar DNA bands were markedly inhibited after washing with chlorhexidine. These results suggest that essential oil and povidone-iodine mouthwash have little effect on oral nitrate-reducing activity. Salivary nitrite production was not reduced by chlorhexidine, but the fainter band of V. dispar DNA suggests that longer daily use might blunt this nitrate-reducing activity.

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Comparison of DNA polymerases for improved forensic analysis of challenging samples

Cited by 16 publications

References 17 publications

Salivary nitrate-nitrite conversion capacity after nitrate ingestion and incidence of <i>Veillonella</i> spp. in elderly individuals

Salivary nitrate-nitrite conversion capacity after nitrate ingestion and incidence of <i>Veillonella</i> spp. in elderly individuals

A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils

The effects of essential oil, povidone-iodine, and chlorhexidine mouthwash on salivary nitrate/nitrite and nitrate-reducing bacteria

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