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Measurement of norovirus

Series: Ernestly measurement

Measurement of norovirus

You may have a sudden stomach ache in the middle winter.
You think of what you ate the day before, pour thinned hypochlorous acid in a spray bottle, and occupy the restroom for a long time.

According to the “2017 food poisoning report” issued by the Japanese Ministry of Health, Labour and Welfare, 20% of food poisoning was caused by norovirus when the cases are sorted by cause. It was the second leading cause with over 200 reported cases that year. Every year, the primary cause of food poisoning is either norovirus or campylobacter.
We examined norovirus using a mobile PCR device.

Sample

Synthesized RNA of Norovirus GII (ATCC: VR-3235SD)

RT enzyme

THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO)

Reaction composition
  μL Remarks (final concentration)
RT Enzyme Mix 0.5 0.5μL/reaction
DNA Polymerase 1 1.0μL/reaction
2x Reaction Buffer 8.5 1x
COG2F forward primer 0.136 800nM
ALPF forward primer 0.136 800nM
COG2R reverse primer 0.136 800nM
RING2AL-TP probe 0.34 200nM
RING1-TP(b) probe (FAM added) 0.255 150nM
Sample 1  
PCR grade water 5  

Reaction sample total 17μL

Program
Process Temperature Time  
Reverse transcription 42℃ 200sec  
Initial denaturation 95℃ 15sec  
Annealing/extension 55℃ 10sec 50cycle
Denaturation 95℃ 3.5sec
Results

In the THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO) we used in the test, the RNA amount (/reaction) was detectable at the range of 4.4 x 105 to 4.4 x 10 copies, demonstrating that a correlation with the Ct value was maintained.

When electrophoresis was performed for 4.4 x 100 copies/reaction in the condition, almost no assumed PCR product was observed and any correlation with the Ct value was poor ⇒ almost no expected PCR product was observed and correlation with the Ct value was poor As a result, we determined that the results were not valid.

It is difficult to evaluate kits for RT made by various companies as conditions were different.
However, it can be said that excellent results were provided from a viewpoint of sensitivity in the conditions listed for the kit.