Detection of bacteria around us by direct PCR
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Bacteria around us can be detected and even identified by polymerase chain reaction or PCR. PCR with primers for bacterial 16S rRNA gene and/or species-specific gene sequences is a useful method in detection of bacteria. Direct PCR offers convenience, speed, and simplicity in this approach because it circumvents the need for bacterial genomic DNA purification from samples. Here, we show detection of the following bacterial species from five sample types, using Invitrogen Platinum Direct PCR Universal Master Mix. • Any bacterium and Escherichia coli on produce • Any bacterium and Escherichia coli on hands • Any bacterium and Escherichia coli in saliva • Any bacterium, Bifidobacterium, and Streptococcus salivarius from buccal swabs • Any bacterium in lake water Find out more about Platinum Direct PCR Universal Master Mix at thermofisher.com/platinum-direct-pcr Discover other PCR enzymes suitable for bacterial detection at thermofisher.com/broad-range-pcr Learn more about PCR at thermofisher.com/pcreducation
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Detection of bacteria around us by direct PCR
- 1. The world leader in serving science Detection of Bacteria with Platinum Direct PCR Universal Master Mix For Research Use Only. Not for use in diagnostic procedures.
- 2. 2 What Is Invitrogen™ Platinum™ Direct PCR Universal Master Mix? thermofisher.com/platinum-direct-pcr A PCR master mix that offers: • Direct amplification from a variety of samples – DNA purification not required prior to PCR • Universal annealing temperature for different primer sets • Co-cycling of targets of different lengths • High yield, specificity, and sensitivity • Efficient amplification of GC-rich sequences • Multiplex PCR of up to 5 targets
- 3. 3 Master Mix Offers Two Protocols for Convenience • Quick • Minimal steps • Sample size critical • Flexible • Amenable to sample size variations • Sample storage options at 4C or –20C Direct protocol Lysis protocol + primers or
- 4. 4 Bacteria Around Us and Their Detection by Direct PCR Fruits/vegetables Any bacterium, Escherichia coli Hand swabs Any bacterium, E. coli Saliva Any bacterium, E. coli Buccal swabs Any bacterium, Bifidobacterium, Streptococcus salivarius Lake water Any bacterium PCR with primers for bacterial 16S rRNA gene and species-specific gene sequences Liquid samples (saliva, lake water) followed the direct approach • 1–5 μL added directly to the 50 μL PCR master mix Swabs (from produce, hands, and mouth) followed the lysis approach • Sample swiped with swab that had been soaked in the lysis buffer (included in the kit) • Swab dipped again in the lysis buffer and drained; 5 μL of the buffer is then added to 50 μL master mix
- 5. 5 Bacteria on Fruits/Vegetables Lower bacterial loads were detected by direct PCR after samples were washed. Apple Nectarine Tomato Bell pepper NTC U W U W U W U W Bacterial 16S rRNA gene sequence E. coli gyrB gene sequence M M Detection of bacteria on fruits and vegetables by direct DNA amplification • 0.45 kb bacterial 16S rRNA sequence • 0.62 kb E. coli gyrB sequence • Direct PCR with the lysis protocol of Platinum Direct PCR Universal Master Mix • Swabs from unwashed (U) and washed (W) samples • M = Thermo Scientific™ GeneRuler™ 1 kb DNA Ladder, NTC = no-template control (swab without sample)
- 6. 6 Bacteria on Hands After Touching Surfaces Less E. coli was detected by direct PCR on hands that had been washed after touching bathroom surfaces. Detection of bacteria on hands by direct DNA amplification • 0.45 kb bacterial 16S rRNA sequence • 0.62 kb E. coli gyrB sequence • Direct PCR with the lysis protocol of Platinum Direct PCR Universal Master Mix • Swabs from hands that had touched bathroom surfaces (+WC) or not (–WC) • M = GeneRuler 1 kb DNA Ladder, NTC = no-template control (swab without sample) Bacterial 16S rRNA gene sequence E. coli GyrB gene sequence +WC –WC Unwashed Washed NTCM Bacterial 16S rRNA gene sequence E. coli gyrB gene sequence M
- 7. 7 Detection of bacteria in human saliva by direct DNA amplification Resident Bacteria in Saliva Bacteria (may have been dead or alive) but no E. coli (negative control) in saliva were detected by direct PCR. • 0.45 kb bacterial 16S rRNA sequence • 0.62 kb E. coli gyrB sequence • Direct PCR with the direct protocol of Platinum Direct PCR Universal Master Mix • Saliva from individuals who had used mouthwash or not • M = GeneRuler 1 kb DNA Ladder, NTC = no-template control (nuclease-free water) Bacterial 16S rRNA gene sequence E. coli gyrB gene sequence NTC –Mouth wash +Mouth wash M
- 8. 8 Indigenous Bacteria in Mouth Indigenous bacterial species (may have been dead or alive) of the mouth were detected by direct PCR. Detection of bacteria from buccal swabs by direct DNA amplification • 0.45 kb bacterial 16S rRNA sequence • 0.55–0.56 kb Bifidobacterium 16S rRNA sequence • 0.5 kb S. salivarius dex sequence • Direct PCR with the lysis protocol of Platinum Direct PCR Universal Master Mix • Buccal swabs from individuals who had used mouthwash or not • M = GeneRuler 1 kb DNA Ladder, NTC = no-template control (swab without sample) NTC –Mouth wash +Mouth wash M Bacterial 16S rRNA gene sequence S. salivarius dextranse gene sequence Bifidobacterium 16S rRNA gene sequence M
- 9. 9 Resident Bacteria in Lake Water Bacteria in lake water were detected by direct PCR. Detection of bacteria in lake water by direct DNA amplification • 0.45 kb bacterial 16S rRNA sequence • Direct PCR with the direct protocol of Platinum Direct PCR Universal Master Mix • Water from three freshwater lakes • M = GeneRuler 1 kb DNA Ladder, NTC = no-template control (nuclease-free water) 5 μL water sample Lake 1 Lake 2 Lake 3 NTC Bacterial 16S rRNA gene sequence M
- 10. 10 Summary Species-specific PCR is a useful method to detect bacteria. Direct PCR offers convenience and speed by circumventing the need for DNA purification from samples. Platinum Direct PCR Universal Master Mix can be used for species-specific PCR with a variety of samples.
- 11. 11 Ordering Information Request a sample* at thermofisher.com/platinum-direct-pcr Description Size Cat. No. Platinum Direct PCR Universal Master Mix 100 reactions A44647100 500 reactions A44647500 4 x 500 reactions A44647200 * Terms and conditions apply.
- 12. The world leader in serving science Thank you For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL111193 0220