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Bacterial Detection Direct PCR Data

The document discusses a PCR master mix called Platinum Direct PCR Universal Master Mix that allows direct amplification from samples without DNA purification. It describes the two protocols and provides examples detecting various bacteria directly from fruits, hands, saliva, mouth and lake water using species-specific PCR primers.

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89 views12 pages

Bacterial Detection Direct PCR Data

The document discusses a PCR master mix called Platinum Direct PCR Universal Master Mix that allows direct amplification from samples without DNA purification. It describes the two protocols and provides examples detecting various bacteria directly from fruits, hands, saliva, mouth and lake water using species-specific PCR primers.

Uploaded by

chiralic
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Detection of Bacteria with Platinum

Direct PCR Universal Master Mix

For Research Use Only. Not for use in diagnostic procedures.

The world leader in serving science


What Is Invitrogen™ Platinum™ Direct PCR Universal Master Mix?

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 thermofisher.com/platinum-direct-pcr


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

2
Master Mix Offers Two Protocols for Convenience

Direct protocol

• Quick

• Minimal steps

• Sample size critical

Lysis protocol

• Flexible

• Amenable to sample size variations


+ primers
• Sample storage options at 4C
or –20C

or

3
Bacteria Around Us and Their Detection by Direct PCR

PCR with primers for bacterial 16S rRNA


Fruits/vegetables
gene and species-specific gene sequences
Any bacterium, Escherichia coli

Hand swabs Liquid samples (saliva, lake water)


Any bacterium, E. coli followed the direct approach

• 1–5 μL added directly to the 50 μL PCR


master mix
Saliva
Any bacterium, E. coli
Swabs (from produce, hands, and mouth)
followed the lysis approach
Buccal swabs
Any bacterium, Bifidobacterium, Streptococcus salivarius • Sample swiped with swab that had been
soaked in the lysis buffer (included in the kit)

Lake water • Swab dipped again in the lysis buffer and


drained; 5 μL of the buffer is then added to
Any bacterium
50 μL master mix

4
Bacteria on Fruits/Vegetables

Apple Nectarine Tomato Bell pepper

M NTC U W U W U W U W M
Detection of bacteria on fruits and
Bacterial 16S rRNA gene sequence 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


E. coli gyrB gene sequence 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)

Lower bacterial loads were detected by direct PCR after samples were washed.

5
Bacteria on Hands After Touching Surfaces
+WC –WC

M Unwashed Washed NTC M

Bacterial 16S rRNA


Bacterial 16S gene
rRNAsequence
gene sequence
Detection of bacteria on hands by direct
DNA amplification

• 0.45 kb bacterial 16S rRNA sequence


• 0.62 kb E. coli gyrB sequence
E.GyrB
E. coli coli gene
gyrB sequence
gene 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)

Less E. coli was detected by direct PCR on hands that had been washed after touching bathroom surfaces.

6
Resident Bacteria in Saliva

–Mouth +Mouth
M NTC
wash wash

Bacterial 16S rRNA gene sequence


Detection of bacteria in human saliva by direct
DNA amplification

• 0.45 kb bacterial 16S rRNA sequence


• 0.62 kb E. coli gyrB sequence

E. coli gyrB gene 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)

Bacteria (may have been dead or alive) but no E. coli (negative control) in saliva were detected by direct PCR.

7
Indigenous Bacteria in Mouth

–Mouth +Mouth
M NTC M
wash wash

Bacterial 16S rRNA gene sequence


Detection of bacteria from buccal swabs by direct
DNA amplification

• 0.45 kb bacterial 16S rRNA sequence


Bifidobacterium 16S
rRNA gene 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
S. salivarius dextranse
gene sequence
• Buccal swabs from individuals who had used mouthwash or not
• M = GeneRuler 1 kb DNA Ladder, NTC = no-template control
(swab without sample)

Indigenous bacterial species (may have been dead or alive) of the mouth were detected by direct PCR.

8
Resident Bacteria in Lake Water

5 μL water sample
M Lake 1 Lake 2 Lake 3 NTC Detection of bacteria in lake water by direct
DNA amplification
Bacterial 16S rRNA gene sequence

• 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)

Bacteria in lake water were detected by direct PCR.

9
Summary

Species-specific PCR Direct PCR offers convenience Platinum Direct PCR Universal
is a useful method to and speed by circumventing Master Mix can be used for
detect bacteria. the need for DNA purification species-specific PCR with a
from samples. variety of samples.

10
Ordering Information

Description Size Cat. No.

100 reactions A44647100

Platinum Direct PCR


Universal Master Mix 500 reactions A44647500

4 x 500
A44647200
reactions

Request a sample* at thermofisher.com/platinum-direct-pcr


* Terms and conditions apply.

11
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

The world leader in serving science

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