Description

Incidence of food poisonings caused by Campylobacter is becoming more frequent than ever in recent years. Campylobacter species are gram-negative, spiral-shaped bacteria that multiply under 3 - 10% oxygen microaerophilic condition. The main symptom caused by Campylobacter is enteritis. And about 90% of the pathogenic bacteria are Campylobacter jejuni (C. jejuni) and a few percent of those are Campylobacter coli (C. coli). Campylobacter fetus (C. fetus) is isolated from specimen of septicemia or meningitidis, and considered as one of pathogenic bacteria for such symptoms. As the symptoms are different according to the bacteria species, it is very important not only to detect presence of the bacteria but also to identify species of those. Currently, detection and identification of Campylobacter is done by culture method. Because the growth of those bacteria is slow, it would take at least 2 days for pre-culture of the bacteria and several days for identification test of bacteria species.Thus the period of about a week is required for certainly identification of Campylobacter species. It have been expected to develop rapid detection and identification method of Campylobacter applying gene detection technology such as PCR.
This kit is designed to specifically and rapidly detect and identify 3 kinds of bacterium species, C. jejuni, C. coli, and C. fetus, by PCR using B subunit gene (cdtB gene) or C subunit gene (cdtC gene) of the Cytolethal distending toxin (cdt genes) as targets. In a Campylobacter species, there are infrequently strains which have deletion or mutation on cdtB gene or cdtC gene. To increase detection precision, detection of both cdtB and cdtC genes will be performed. This kit allows to detect the target genes in highly sensitive because this kit contains enzyme for hot start PCR, TaKaRa Ex Taq™ Hot Start Version, for PCR amplification, by precluding miss-priming during preparation of reaction solution and non-specific amplification derived from primer dimmer.

This kit is designed by TaKaRa through kindly cooperation with Osaka Prefecture University and Fuso Pharmaceutical Industries, Ltd.

Components

(for 100 reactions^*1, 50 µl PCR)

1. Premix Ex Taq™ HS*2 (2×conc.)	500 µl×5
2. cdtB Primer Mix	250 µl
3. cdtC Primer Mix	250 µl
4. dH2O	1 ml×3
5. C. jejuni Positive Control	150 µl
6. C. coli Positive Control	150 µl
7. C. fetus Positive Control	150 µl

*1 50 reactions for cdtB gene detection and 50 reactions for cdtC gene detection.
*2 2×concentration of PCR reaction reagent which contains TaKaRa Ex Taq™ HS, Reaction Buffer, and dNTP mixture. The activity might reduce by repeating the freeze and thaw process, thus divide 25 µl of it in PCR tube and store at -20°C after thawed out.

Storage

-20°C

Reagents and instruments not supplied in the kit

[ Reagents ]

1. Sterilized distilled water

2. NuSieve^® 3 : 1 Agarose (Lonza Rockland. Inc.)

3. Electrophoresis buffer (TBE powder (Cat. #T905) or 20×TAE Buffer (Lonza Rockland. Inc.))

4. DNA marker [100bp DNA ladder (Cat. #3407A/B) ]

5. Loading buffer (6× : 36% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 30 mM EDTA) (this loading buffer is supplied with DNA marker listed on 4)

6. DNA stain reagents [SYBR^® Green I (Lonza Rockland. Inc.) or Ethidium Bromide]

[ Instruments ]

1. Heating block (applicable at 95°C)

2. Refrigerated centrifuge, compatible with 1.5ml tubes

3. Thermal Cycler for PCR
      TaKaRa PCR Thermal Cycler Dice™ (Cat. #TP600/TP650)
      TaKaRa PCR Thermal Cycler Dice™ mini (Cat. #TP100)

4. Electrophoresis apparatus
      Mupid^®-2plus (Cat. #AD110)
      Mupid^®-exU (Cat. #AD140)

5. Equipment to photograph electrophoresis gel

[ Others ]

1. 0.2 ml PCR tube [ 0.2 ml Hi-Tube Dome Cap (Cat. #NJ200) , TaKaRa Micro PCR Tube (Cat. #9047) ]

2. Micropipette for 200 µl, 20 µl, and 10 µl

3. Micropipette tips (with hydrophobic filter)