Integrated fluidic chip for identification of fruit fly type and use thereof

Abstract

The invention discloses an integrated fluidic chip for identification of a fruit fly type and a use thereof. The integrated microfluidic chip comprises specific primers for fruit fly type identification and a TaqMan-MGB probe. The invention provides a fruit fly type molecular identification technology utilizing the integrated fluidic chip. A test proves that the integrated microfluidic chip-based molecular identification technology realizes fast identification of a fruit fly type, has good specificity, high flux, a low cost and simple processes, realizes identification of a type of non-adult fruit fly or fruit fly adult residual body, provides a fast identification tool and technology for import and export plant quarantine and nationwide fruit fly epidemic monitoring, effectively prevents invasion and diffusion of dangerous fruit fly, protects agricultural production and ecological safety and promotes trade and economic development in China.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an integrated flow chip for identifying fruit fly species and its application. Background technique

[0002] Fruit flies (Fruit Flies) belong to Diptera (Diptera) Tephritidae (Tephritidae), widely distributed all over the world, mostly occur in tropical and subtropical regions. There are about 500 genera and 4,500 species of Tephritidae, and there are more than 250 species of fruit flies with important economic significance. Some fruit flies can cause devastating disasters to fruit production. There are five genera of fruit flies of economic importance in the world, namely Anastrepha, Bactrocera, Ceratitis, Dacus and Around the genus Rhagoletis.

[0003] In my country's current list of imported plant quarantine pests, the quarantine fruit flies include Anastrepha, Bactrocera, Bactrocera spp. species), Bactrocera eufa, Bactrocera cerevisiae, Bactrocera dorsalis, Bactroce...

Examples

Embodiment 1

[0130] Embodiment 1, identification of 27 kinds of fruit fly specific primer pairs and the acquisition of probes

[0131] 1. Acquisition of the fruit fly mtDNA COI gene sequence

[0132] 1. The fruit fly samples to be tested are shown in Table 1. The samples were all placed in absolute ethanol and stored at -20°C for later use.

[0133] Table 1. Types of fruit fly samples tested and information on collection places

[0134]

[0135]

[0136]

[0137]

[0138] 2. Use the "Blood / Cell / Tissue Genomic DNA Extraction Kit" (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of each fruit fly sample in Table 1 (most samples are extracted from a single foot, and a few The sample is extracted from a single head), and the specific operation process refers to the instructions to obtain the genomic DNA of each fruit fly sample in Table 1.

[0139] The genomic DNA of each fruit fly sample in Table 1 was used as a template, and the universal primer L...

Embodiment 2

[0150] Example 2, Preparation of integrated flow chip for identification of 27 species of fruit flies and its specificity detection

[0151] 1. Preparation of integrated flow chip

[0152] 1. Initialization of 48.48 dynamic chips

[0153] (1) Open the package of the chip and use it within 24 hours;

[0154] (2) Inject the Control Line Fluid into the collectors at both ends of the 48.48 dynamic chip, and be careful not to drop the Control Line Fluid into the Assay Inlets or Sample Inlets;

[0155] (3) Remove the blue protective film on the bottom of the IFC chip;

[0156] (4) Transfer the chip to the IFC Controller, run the Primer (113x) program to initialize the chip, and the initialization time is 10 minutes; after the initialization is completed, the sample loading operation is completed within 60 minutes.

[0157] 2. Preparation of standard reaction system and sample mixture

[0158] (1) According to the test system shown in Table 3, prepare 10×Assays of 27 kinds of fru...

Embodiment 3

[0210] Example 3, the application of integrated flow chip in the identification of fruit flies

[0211] 1. Fruit fly samples to be tested

[0212] Taking the following non-adult fruit fly sample as an example, the integrated flow chip described in Example 2 was used to identify its species.

[0213] In May 2012, Beijing Entry-Exit Inspection and Quarantine Bureau intercepted 3 fruit fly larvae from lotus mist imported from Thailand;

[0214] In May 2014, Beijing Entry-Exit Inspection and Quarantine Bureau intercepted 2 fruit fly larvae from mangoes imported from Tanzania;

[0215] In July 2009, two fruit fly larvae were collected from the rotten fruits of bitter gourd in Nanning, Guangxi Zhuang Autonomous Region, my country;

[0216] In October 2011, two fruit fly pupae whose host was citrus were submitted to the Plant Protection and Inspection Station of Chengdu City, Sichuan Province, my country;

[0217] In August 2011, 3 capsicum fruit fly eggs were raised in the Nation...

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an integrated flow chip for identifying fruit fly species and its application. Background technique

[0002] Fruit flies (Fruit Flies) belong to Diptera (Diptera) Tephritidae (Tephritidae), widely distributed all over the world, mostly occur in tropical and subtropical regions. There are about 500 genera and 4,500 species of Tephritidae, and there are more than 250 species of fruit flies with important economic significance. Some fruit flies can cause devastating disasters to fruit production. There are five genera of fruit flies of economic importance in the world, namely Anastrepha, Bactrocera, Ceratitis, Dacus and Around the genus Rhagoletis.

[0003] In my country's current list of imported plant quarantine pests, the quarantine fruit flies include Anastrepha, Bactrocera, Bactrocera spp. species), Bactrocera eufa, Bactrocera cerevisiae, Bactrocera dorsalis, Bactroce...

Examples

Embodiment 1

[0130] Embodiment 1, identification of 27 kinds of fruit fly specific primer pairs and the acquisition of probes

[0131] 1. Acquisition of the fruit fly mtDNA COI gene sequence

[0132] 1. The fruit fly samples to be tested are shown in Table 1. The samples were all placed in absolute ethanol and stored at -20°C for later use.

[0133] Table 1. Types of fruit fly samples tested and information on collection places

[0134]

[0135]

[0136]

[0137]

[0138] 2. Use the "Blood / Cell / Tissue Genomic DNA Extraction Kit" (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of each fruit fly sample in Table 1 (most samples are extracted from a single foot, and a few The sample is extracted from a single head), and the specific operation process refers to the instructions to obtain the genomic DNA of each fruit fly sample in Table 1.

[0139] The genomic DNA of each fruit fly sample in Table 1 was used as a template, and the universal primer L...

Embodiment 2

[0150] Example 2, Preparation of integrated flow chip for identification of 27 species of fruit flies and its specificity detection

[0151] 1. Preparation of integrated flow chip

[0152] 1. Initialization of 48.48 dynamic chips

[0153] (1) Open the package of the chip and use it within 24 hours;

[0154] (2) Inject the Control Line Fluid into the collectors at both ends of the 48.48 dynamic chip, and be careful not to drop the Control Line Fluid into the Assay Inlets or Sample Inlets;

[0155] (3) Remove the blue protective film on the bottom of the IFC chip;

[0156] (4) Transfer the chip to the IFC Controller, run the Primer (113x) program to initialize the chip, and the initialization time is 10 minutes; after the initialization is completed, the sample loading operation is completed within 60 minutes.

[0157] 2. Preparation of standard reaction system and sample mixture

[0158] (1) According to the test system shown in Table 3, prepare 10×Assays of 27 kinds of fru...

Embodiment 3

[0210] Example 3, the application of integrated flow chip in the identification of fruit flies

[0211] 1. Fruit fly samples to be tested

[0212] Taking the following non-adult fruit fly sample as an example, the integrated flow chip described in Example 2 was used to identify its species.

[0213] In May 2012, Beijing Entry-Exit Inspection and Quarantine Bureau intercepted 3 fruit fly larvae from lotus mist imported from Thailand;

[0214] In May 2014, Beijing Entry-Exit Inspection and Quarantine Bureau intercepted 2 fruit fly larvae from mangoes imported from Tanzania;

[0215] In July 2009, two fruit fly larvae were collected from the rotten fruits of bitter gourd in Nanning, Guangxi Zhuang Autonomous Region, my country;

[0216] In October 2011, two fruit fly pupae whose host was citrus were submitted to the Plant Protection and Inspection Station of Chengdu City, Sichuan Province, my country;

[0217] In August 2011, 3 capsicum fruit fly eggs were raised in the Nation...

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