Page 13 - Microorganism Species Analysis

Norovirus Detection Reagent Kit Norovirus Detection Reagent Kit
Nucleic Acid (RNA)
Microorganism Solutions Microorganism Solutions

Direct Detection of Norovirus in Feces Using RT-PCR with the Norovirus Data
G1/G2 Detection Reagent Kit
Noroviruses are a cause of food poisoning that can be directly transmitted from the feces or vomit of an infected person, as Detection of Norovirus in Feces Using the Norovirus G1/G2 Detection Reagent Kit
well as from foods or drinks containing the virus. The 2008 "Hygiene Control Manual for Commercial Kitchens" (No. 0618005 Microorganism Species Observation of
issued by Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare) prescribes a test for norovirus Test flow I.C. Electrophoresis Specific amplification product length: 142 bp
Tm analysis Tm peak temperature: 87 °C ± 1 °C
as part of the stool examinations for cooking staff, where required. 1. Treat four each of eight stool samples according to G1 Yes No
Shimadzu developed the Norovirus G1/G2 Detection Reagent Kit to directly amplify and detect two genogroups (G1/G2) the spiking protocol with the G1 and G2 Norovirus Electrophoresis Specific amplification Yes Positive Positive
Observation of
product length: 86 bp
from stool samples. It permits direct detection of viruses in feces without the need to purify a template gene. Detection Reagent Kit, respectively, and then
Tm analysis Tm peak temperature: No Negative Indeterminate
Microorganism Species
subject them to the reverse transcriptase reaction 83 °C ± 1 °C
and PCR amplification. Electrophoresis Specific amplification product length: 205 bp
I.C. Tm analysis Tm peak temperature: 89 °C ± 1 °C
2. Detect the amplified genes and evaluate G1/G2 G2 Yes No
Features of the Norovirus G1/G2 Detection Reagent Kit type using the melting temperature (Tm) analysis Electrophoresis Specific amplification Yes Positive Positive
product length: 98 bp
method by agarose gel electrophoresis or
Tm analysis Tm peak temperature:
Simply spike the reagent in the reaction tube; it is not necessary to remove the sample from the reaction tube. real-time PCR. 85 °C ± 1 °C No Negative Indeterminate
Eliminates the need for tedious RNA extraction and purification from stool samples and significantly reduces the cost
G1/G2 Kit Evaluation Criteria
and time required for sample pretreatment. Microorganism Species Identification of
Mix centrifuge supernatant from the fecal suspension with the sample processing reagent and incubate for one hour
at 85 °C to cause a direct reverse transcriptase reaction and PCR amplification, unaffected by RNA catabolic Electrophoresis analysis Melting temperature (Tm) analysis
enzymes or PCR amplification inhibitors. M: molecular marker M1 2 3 4 G1 I.C.* : Stool sample 1
Identification of
All stages from stool sample pretreatment to amplification are performed in a single tube, making the process (øX174 DNA Hinc 2 digest) : Stool sample 2 Evaluation results
: Stool sample 3
Microorganism Species
Detection with 1: Stool sample 1 -∆ (fluorescence intensity) /∆ (temperature) : Stool sample 4 Stool sample 1: G1 positive
suitable for processing large volumes. G1 kit 2: Stool sample 2 Stool sample 2: G1 positive
3: Stool sample 3
Stool sample 3: G1 negative
The kit reagents are spiked with internal control DNA to prevent false negatives. 4: Stool sample 4 I.C.* Stool sample 4: G1 negative
G1 75 80 85 90 95
A high correlation has been confirmed with the Japanese Ministry of Health, Labour and Welfare method, which is Temperature (°C)
the standard method for norovirus detection and inspection. M 5 6 7 8 G2 I.C.* : Stool sample 5
M: molecular marker : Stool sample 6
Combination with the MCE-202 MultiNA Microchip Electrophoresis System for DNA/RNA analysis can automate DNA (øX174 DNA Hinc 2 digest) : Stool sample 7 Evaluation results
Detection with 5: Stool sample 5 : Stool sample 8 Stool sample 5: G2 positive
analysis by agarose gel electrophoresis after PCR amplification. 6: Stool sample 6 -∆ (fluorescence intensity) /∆ (temperature) Stool sample 6: G2 positive
G2 kit 7: Stool sample 7 Stool sample 7: G2 negative
8: Stool sample 8 I.C.* Stool sample 8: G2 negative
G2 75 80 85 90 95 Specific Microorganisms Detection of
Temperature (°C) *Internal control
Detection of
Fig. 1 Detection of Norovirus in Feces Using the Norovirus G1/G2 Detection Reagent Kit
(Agarose Gel Electrophoresis and Melting Temperature Tm Analysis)
Specific Microorganisms
19 µL sample RT-PCR reaction solution
processing (Example using Ampdirect ® technology) Both agarose gel electrophoresis analysis and melting temperature (Tm) analysis were able to clearly detect the
1 µL 5% to 10% fecal reagent Direct spiking of sample after pretreatment
suspension centrifuge absence or presence of norovirus and evaluate the G1/G2 type in all samples.
supernatant
RNase inactivation RNA amplification reaction
RNA extraction (RT-PCR) in 2 steps in 1 tube
from virus Time required: approx.
85 °C, 1 minute 3 hours Detection of Norovirus in Feces Using MultiNA
Time required: 3 minutes per sample Both norovirus-derived peaks (86 bp or 98 bp) and peaks derived from the internal control (142 bp or 205 bp) were Components Analysis of Microorganism-Derived
detected from G1/G2 positive samples.
The Norovirus Detection Reagent Kit significantly reduces
the time for sample pretreatment by eliminating the need MultiNA achieves superior separation to agarose gel electrophoresis for PCR amplification fragments up to 100 base pairs.
Components
Norovirus G1/G2 Detection Reagent Kit for RNA extraction and purification.
Fig. 1 shows the results of analyzing amplification products in G1/G2 positive samples using MultiNA with the
DNA-500 kit.
Analysis of Microorganism-Derived
MultiNA can perform high-speed, automated analysis of up to 120 samples at equivalent or less cost than agarose gel
electrophoresis.
98 bp
205 bp (IC) Other Applications
Norovirus_G2+
86 bp
142 bp (IC)
Norovirus_G1+
25 bp DNA Ladder
Other Applications
MCE-202 MultiNA
Microchip Electrophoresis System for DNA/RNA Analysis Fig. 1 MultiNA Detection of Norovirus in Feces Using the Norovirus G1/G2 Detection Reagent Kit
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