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.
German cockroach limb, German cockroach feces
Calibration curve measurement: Genes were extracted from limbs using GenCheck DNA Extraction Reagent (FASMAC Co., Ltd.). Measurement of feces: PBS(-) stirring dilution
|μL||Remarks (final concentration)|
|Enzyme: KAPA 3G Plant (2.5 U/μL)||0.6||1.5U/reaction|
|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|
|PCR grade water||5.8|
Reaction sample total 17μL
[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.