Specific primer, probe, kit and method for detecting barley component

Abstract

The invention provides a specific primer, a probe, a kit and a method for detecting barley component. A large number of primers are designed based on FDH, GLU2a, RBCL, ACPS and UDPg genes of barley, and primers and probes with species specificity are screened out through a large number of primer screening researches. When the primers and the probes are used for detecting barley component in a sample to be detected, barley and other species adulterants can be accurately separated, the absolute detection sensitivity reaches 0.2 copies / microliter, the relative sensitivity can reach 0.5%, and theidentification result is accurate, stable and reliable.

Description

Technical field

[0001] The invention belongs to the technical field of molecular biology detection, and specifically relates to a specific primer, probe, kit and method for detecting barley components. Background technique

[0002] With a large number of researches on genetically modified products and their widespread application in daily life, the safety issues that genetically modified products bring to human health and the living environment have aroused widespread concern and debate around the world. Therefore, while actively researching genetically modified technology, countries around the world have also conducted a lot of research on the detection and analysis methods of various genetically modified foods. However, due to the large variety and quantity of genetically modified foods, especially after many genetically modified foods are processed and stored under various conditions, the genetically modified ingredients are largely degraded, making detection more difficult. ...

Examples

Embodiment 2

[0061] Example 2 Probe

[0062] This example provides a probe for detecting the components of barley. The probe designed for the ACPS gene of barley is as follows, see SEQ ID NO: 3 in the sequence table: the sequence of the probe is: 5'-TCGCGCCATTCAAGCCGTGATACGTC -3'.

Embodiment 3

[0063] Example 3 Common PCR detection kit

[0064] This embodiment provides a kit for detecting barley components, each of the kits includes a primer solution part and a PCR reaction solution part that are individually packaged; the primer solution part contains the components in Example 1 The specific primers; the PCR reaction solution part contains amplification buffer for PCR amplification, dNTPs, Taq DNA polymerase and sterile ultrapure water, the formula of each kit is:

[0065] Upstream primer, 10μM, 0.5~1.5μL;

[0066] Downstream primer, 10μM, 0.5~1.5μL;

[0067] Taq DNA polymerase, 5U / μL, 0.2~1.2μL;

[0068] 10×Taq DNA polymerase buffer (containing 10mmol / L Mg 2+ ), 2.5μL;

[0069] dNTPs, 2.5mM, 0.25-1μL; add sterile double-distilled water to a total volume of 25μL.

Embodiment 4

[0070] Example 4 Real-time fluorescent PCR detection kit

[0071] This embodiment provides a kit for detecting barley components, each of the kits includes a primer solution part and a PCR reaction solution part that are individually packaged; the primer solution part contains the components in Example 1 The specific primers and the probe described in Example 2; the PCR reaction solution part contains 2×TaqMan Universal PCR Master Mix containing dNTPs, Taq DNA polymerase and sterile ultra Pure water, the formula of each kit is:

[0072] 2×TaqMan Universal PCR Master Mix 12.5μL;

[0073] Upstream primer (10μM) 1.0μL;

[0074] Downstream primer (10μM) 1.0μL;

[0075] Probe (10μM) 0.5μL;

[0076] Add sterile double-distilled water to a total volume of 25μL.

Technical field

[0001] The invention belongs to the technical field of molecular biology detection, and specifically relates to a specific primer, probe, kit and method for detecting barley components. Background technique

[0002] With a large number of researches on genetically modified products and their widespread application in daily life, the safety issues that genetically modified products bring to human health and the living environment have aroused widespread concern and debate around the world. Therefore, while actively researching genetically modified technology, countries around the world have also conducted a lot of research on the detection and analysis methods of various genetically modified foods. However, due to the large variety and quantity of genetically modified foods, especially after many genetically modified foods are processed and stored under various conditions, the genetically modified ingredients are largely degraded, making detection more difficult. ...

Examples

Embodiment 2

[0061] Example 2 Probe

[0062] This example provides a probe for detecting the components of barley. The probe designed for the ACPS gene of barley is as follows, see SEQ ID NO: 3 in the sequence table: the sequence of the probe is: 5'-TCGCGCCATTCAAGCCGTGATACGTC -3'.

Embodiment 3

[0063] Example 3 Common PCR detection kit

[0064] This embodiment provides a kit for detecting barley components, each of the kits includes a primer solution part and a PCR reaction solution part that are individually packaged; the primer solution part contains the components in Example 1 The specific primers; the PCR reaction solution part contains amplification buffer for PCR amplification, dNTPs, Taq DNA polymerase and sterile ultrapure water, the formula of each kit is:

[0065] Upstream primer, 10μM, 0.5~1.5μL;

[0066] Downstream primer, 10μM, 0.5~1.5μL;

[0067] Taq DNA polymerase, 5U / μL, 0.2~1.2μL;

[0068] 10×Taq DNA polymerase buffer (containing 10mmol / L Mg 2+ ), 2.5μL;

[0069] dNTPs, 2.5mM, 0.25-1μL; add sterile double-distilled water to a total volume of 25μL.

Embodiment 4

[0070] Example 4 Real-time fluorescent PCR detection kit

[0071] This embodiment provides a kit for detecting barley components, each of the kits includes a primer solution part and a PCR reaction solution part that are individually packaged; the primer solution part contains the components in Example 1 The specific primers and the probe described in Example 2; the PCR reaction solution part contains 2×TaqMan Universal PCR Master Mix containing dNTPs, Taq DNA polymerase and sterile ultra Pure water, the formula of each kit is:

[0072] 2×TaqMan Universal PCR Master Mix 12.5μL;

[0073] Upstream primer (10μM) 1.0μL;

[0074] Downstream primer (10μM) 1.0μL;

[0075] Probe (10μM) 0.5μL;

[0076] Add sterile double-distilled water to a total volume of 25μL.

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