Page 10 - Microorganism Species Analysis

Ampdirect Plus Ampdirect Plus
Nucleic Acid (DNA)
Microorganism Solutions Microorganism Solutions

Detection of Microorganism Genes Using Ampdirect Plus Data
The rapid checking and detection of specific microorganisms, such as bacteria that cause food poisoning, pathogenic
microorganisms, and infectious viruses, is demanded in the fields of foods, pharmaceuticals, and the environment. Detection of Verotoxin-Producing Escherichia coli (O157)
Conventionally, the mainstream testing methods were cultivation testing and methods using the antigen-antibody reaction.
According to the "Hygiene Control Manual for Commercial Kitchens" notified by the Japanese Ministry of Health, Microorganism Species Observation of
Cultivation testing takes a long time and has problems handling microorganisms (such as viruses) that are not easy to
Labour and Welfare, cooking staff in commercial kitchens producing more than 300 meals of the same menu or more
cultivate. In recent years, gene-based testing methods have become more prevalent.
than 750 meals per day must undergo health checks and a stool examination, including a test for enterohemorrhagic
Normally, the PCR amplification of genes requires the extraction and purification of template DNA. However, the
Observation of
E. coli, at least once per month and a norovirus examination between October and March, if necessary.
revolutionary Ampdirect Plus DNA Amplification Reagent developed by Shimadzu eliminates or simplifies this DNA extraction
and purification process to accelerate, simplify, and reduce the cost of gene detection.
Microorganism Species
Test flow
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1. Take 500 µL 10% fecal suspension (containing E. coli O157 10 to 10 CFU/µL) in a microcentrifuge tube.
Features of the Ampdirect Plus Microorganism Gene Detection System 2. Heat treat at 95 °C for 5 minutes and centrifuge for 5 minutes to precipitate out the solids.
3. Perform real-time PCR or normal PCR using 1 µL supernatant as the template to detect the verotoxin gene
Ampdirect Plus eliminates the need for (or simplifies) the extraction and purification of template DNA that is required
(VT1/VT2) -derived DNA fragments.
for PCR amplification.
It reduces inhibition of PCR amplification by proteins, polysaccharides, and other impurities in the sample to achieve Microorganism Species Identification of
stable gene amplification.
It is apparent that the Ampdirect
It saves the cost and time required for the extraction and purification of template DNA and reduces the risk of Real-time PCR Melting curve analysis Electrophoresis DNA Amplification Reagent can be
cross-contamination. VT1 M 1 2 3 4 5 6 7 8 used for the high-sensitivity
Identification of
It can detect microorganisms in all samples, including foods, blood (whole blood, plasma, and serum), urine, PCR Base Line Subtracted CF RFU -d(RFU)/dT 392 bp 349 bp detection of enterohemorrhagic E.
Microorganism Species
340 bp
sputum, throat swab, and feces. coli-derived toxin genes in feces
Combination with the MCE-202 MultiNA Microchip Electrophoresis System for DNA/RNA analysis can automate DNA (10 CFU/mL in supernatant,
-1
analysis by agarose gel electrophoresis after PCR amplification. Cycle Temperature, Celsius 3
equivalent to 10 CFU/mL in culture
M 1 2 3 4 5 6 7 8
PCR Base Line Subtracted CF RFU -d(RFU)/dT 392 bp 404 bp
fluid).
VT2
340 bp
Plants
Tail Specific Microorganisms Detection of
Cycle Temperature, Celsius
Blood/dried blood Microorganisms 1: 10 copies/tube 2: 10 copies/tube 3: 10 copies/tube 4: 10 copies/tube
3
5
1
2
-1
0
Detection of
5: 10 copies/tube 6: 10 copies/tube 7: Negative control (negative feces) 8: Negative control (purified water)
Oral mucosa cells Insects
Fig. 1 Real-Time PCR Amplification Curve Using SYBR Green I (Left),
Specific Microorganisms
Dissociation Curve Analysis (Center), and Verotoxin Gene (VT1/VT2)
Paraffin section -Derived DNA Detection Results Using Electrophoresis (Right) *CFU : Colony Forming Unit
Cultivated cells
PCR
®
Ampdirect Plus
Nova Tad™HS
Primer Detection of Fungi in the Environment
Spike with sample The rapid and simple detection and investigation of microorganisms is demanded due to the recent introduction of
HACCP at the manufacturing stage by food manufacturers. The detection of fungus such as mould in the environment Components Analysis of Microorganism-Derived
Ampdirect Plus Ampdirect Plus offers rapid gene amplification of using Ampdirect is shown below.
DNA Amplification Reagent samples derived from various sources.
Components
Test flow
1. Spike SDS-Proteinese K solution with a trace amount of bacteria and incubate for one hour at 55 °C.
2. Spike the Ampdirect (PCR) reaction solution with the solution above as the template and perform PCR amplification
Analysis of Microorganism-Derived
using a prokaryote/eukaryote common area primer.
It can be seen that Ampdirect is also
Samples
M 1 2 3 4 5 6 7 8 M effective for the amplification of fungi
1: Escherichia coli (404 bp)
2: Staphylococcus aureus (423 bp) observed in the environment.
3: Budding yeast Other Applications
4: Candida The amplified DNA fragments can be
5: Yeast Eukaryote common area subjected to species identification by
6: Black mould from the kitchen
7: Green mould from the fruit sequence decoding or difference
8: Negative control
Other Applications
M: øX174 Hinc II digest distinction based on differences in
MCE-202 MultiNA restriction enzyme fragment lengths
Microchip Electrophoresis Fig. 1 Results of PCR Amplification Using Ampdirect (Agarose Electrophoresis) (RFLP).
System for DNA/RNA Analysis MultiNA Microchips and Reagent Kits
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