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Presence of “German cockroach”

Series: Ernestly measurement

Presence of “German cockroach

It appeared in our house!! It finally appeared. Where do they come from and where do they hide? We examined German cockroaches using a mobile PCR device.

Sample

German cockroach limb, German cockroach feces

Pretreatment

Calibration curve measurement: Genes were extracted from limbs using GenCheck DNA Extraction Reagent (FASMAC Co., Ltd.). Measurement of feces: PBS(-) stirring dilution

Reaction composition
μL Remarks (final concentration)
Enzyme: KAPA 3G Plant (2.5 U/μL) 0.6 1.5U/reaction
Buffer (×2) 8.5 x1
F-Primer: German cockroaches (100 μM) 0.1 600nM
R-Primer: German cockroaches (100 μM) 0.1 600nM
Probe: German cockroaches (100 μM) 0.05 300nM
MgCl2(25mM) 0.85 1.25mM
Sample 1
PCR grade water 5.8

Reaction sample total 17μL

Program
Process Temperature Time
Initial denaturation 95℃ 15sec
Annealing/extension 60℃ 10sec 50cycle
Denaturation 95℃ 3.5sec
Results

[First step: Creation of a calibration curve] Sample: German cockroach limb, Pretreatment: Genes were extracted from limbs using GenCheck DNA Extraction Reagent (FASMAC Co., Ltd.), the DNA amount was calculated using a spectrophotometer (from OD260 value, DNA concentration: 249 μg/mL), serial dilution was performed, and supernatant 1 μL was used as the template to create a calibration curve.

Dilution of extracted DNA Dilution rate Log value Ct value
Undiluted solution (DNA concentration: 249 μg/mL) 0 21.87
Diluted 10 fold with water -1 25.8
Diluted 100 fold with water -2 29.76
Diluted 1,000 fold with water -3 33.3
Diluted 10,000 fold with water -4 37.17
Diluted 100,000 fold with water -5 42.45
Diluted 1,000,000 fold with water -6 No detection

[Second step: Measurement of feces] Sample: German cockroach feces Pretreatment: Feces were collected, 1 to 5 pieces of feces were soaked in PBS(-) 100 μL, and supernatant 1 μL extracted using a vortex mixer for 5 minutes as well as centrifuging at 12,000 rpm for 10 minutes was used as the template for direct PCR measurement.

The study result was presented by NSG Group at the 39th annual conference of the Japanese Society of Food Microbiology. Specimen for this test were provided by Civil International Corporation Co., Ltd.