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Measurement of Campylobacter

Series: Ernestly measurement

Measurement of Campylobacter

According to the “2017 food poisoning report” issued by the Japanese Ministry of Health, Labour and Welfare, 30% of food poisoning was caused by Campylobacter when the cases are sorted by cause. It was the primary cause with over 300 reported cases that year.
We examined Campylobacter using a mobile PCR device.

Sample

C. jejuni culture specimen

Pretreatment

Calibration curve measurement:

  1. 1×106 to 1×107 CFU/mL were inoculated in Preston medium 10ml *1.
  2. They were cultured at 42 degrees Celsius for 24 hours (microaerobic culture).
  3. The cultured suspension was diluted in steps.

Measurement of cultured suspension via direct PCR:

  1. Seed bacteria of three different concentrations were inoculated in Puchitto-Campylo 10 mL.
  2. They were cultured for 6 to 48 hours (microaerobic culture).
  3. Specimens were taken every 2 hours of culture.
Reaction composition
μL Remarks (final concentration)
Enzyme: KAPA 3G Plant (2.5 U/μL) 0.6 1.5U/reaction
Buffer (×2) 8 x1
LightMix Modular CampyloBacter 0.5
MgCl2(25mM) 0.8 1.25mM
Sample 1
PCR grade water 5.1

Reaction sample total 16μL

Program
Process Temperature Time
Initial denaturation 95℃ 15sec
Annealing/extension 60℃ 15sec 50cycle
Denaturation 95℃ 4sec
Results

[First step: Creation of a calibration curve]
Sample
Cultured suspension (direct PCR)
Pretreatment

  1. C. jejuni 1E+06 to 1E+07 CFU/mL were inoculated in Preston medium 10 mL.
  2. They were cultured at 42 degrees Celsius for 24 hours (microaerobic culture).
  3. The cultured suspension was diluted in steps, and 1 μL of supernatant was used as the template to create a calibration curve.

The same sample was cultured in an agar medium and the number of colonies was calculated.

DNA dilution Dilution rate
Log value
Ct value
Undiluted solution (24-hour culture) 0 21.01
Diluted 10 fold with water -1 24.96
Diluted 100 fold with water -2 28.64
Diluted 1,000 fold with water -3 33.22
Diluted 10,000 fold with water -4 36.28
Diluted 100,000 fold with water -5 42.30
Diluted 1,000,000 fold with water -6 Not detected

[Second step: Measurement via direct PCR every 2 hours of culture]
Sample
Cultured suspension (direct PCR)
Pretreatment

  1. Seed bacteria of three different concentrations were inoculated in Preston medium 10 mL.
  2. They were cultured for 6 to 48 hours (microaerobic culture).
  3. Specimens were taken every 2 hours of culture.

※1 Puchitto-Campylo (Preston medium) is a product of Nikken-bio Co., Ltd.
The study result was presented by NSG Group at the 39th annual conference of the Japanese Society of Food Microbiology.
This test was carried out and the results were provided by the NPO Biomedical Science Association.