Detection method for irradiation-related characteristic metabolic markers of bactrocera dorsalis under various storage conditions and application

Abstract

The invention discloses a method for detecting related characteristic metabolic markers after irradiation of bactrocera dorsalis in various storage environments and application of the method. The solid phase microextraction-gas chromatography-mass spectrometry (GC-MS) is used for analyzing the metabolite of the test insect, so that the influence of irradiation treatment on the metabolism of the bactrocera dorsalis is researched, and the method has a very good application prospect. According to the method, five differential metabolites are extracted from the bactrocera dorsalis irradiated in different environments, and the application prospect is wide when the five differential metabolites serve as biomarkers for detecting bactrocera dorsalis irradiation under different storage conditions.

Description

Technical Field

[0001] The invention relates to a technology for detecting irradiation of fruit fly, and in particular to a method for detecting irradiation-related characteristic metabolic markers of fruit fly and an application thereof. Background Art

[0002] With the in-depth development of international trade, the risk of pest spread has increased sharply, seriously threatening local planting and ecological health. It has attracted much attention in the trade of fresh plant products. According to international practice, quarantine treatment is usually required before trade. The common treatment technologies currently used in the trade of fresh plant products include heat treatment, cold treatment, irradiation treatment and methyl bromide fumigation treatment. Heat treatment is only applicable to tropical fruits; cold treatment has a long cycle; methyl bromide is strictly controlled because of its destructive effect on the atmospheric ozone layer; irradiation treatment is fast, efficient and has a wide range of applications. Its application scenarios have become a direction worthy of attention in the field of fresh products. At present, the irradiation treatment technology for fruit flies is relatively mature and complete. The International Plant Protection Convention has promulgated a number of relevant standards. The recommended quarantine irradiation dose can meet the quarantine treatment requirements and make the pests lose their reproductive ability, but it cannot kill the quarantine pests immediately. Therefore, live insects without reproductive ability may still be found in the products after irradiation treatment.

[0003] Studies have shown that some fruit flies will develop a blackening phenomenon on their bodies after irradiation, and this phenomenon can be used to determine whether they have been irradiated. Since morphological changes often vary greatly from individual to individual, a determination method based on enzymatic changes and specific gene expression levels was later developed to determine whether fruit flies have been irradiated under normal temperature and conditions. In order to protect quality, modified atmosphere packaging is often used in the trade of fresh agricultural products. Although it has been verified that low-oxygen and ultra-low-oxygen environments do not affect the treatment effect of quarantine irradiation, the modified atmosphere environment changes the oxidative stress state of fruit flies and changes the expression of related products. Therefore, the application scenarios of existing detection technologies are extremely limited, and a faster and more widely applicable irradiation detection technology needs to be developed. Summary of the invention

[0004] The purpose of the present invention is to provide a detection method and application of irradiation-related characteristic metabolic markers of Bactrocera dorsalis under different storage conditions.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for detecting characteristic metabolic markers related to irradiation of Bactrocera dorsalis, comprising the following steps: (1) Taking a specific pest as the research object, storing it in a low oxygen, ultra-low oxygen and air environment at room temperature, and then irradiating it with the irradiation dose recommended by international plant quarantine measures; (2) Solid phase microextraction-gas chromatography-mass spectrometry (GC-MS) was used to detect the metabolites expressed by pests in different storage environments after irradiation, and characteristic metabolic markers related to controlled atmosphere irradiation were screened out from the differentially expressed metabolites under different storage conditions.

[0006] The pests include but are not limited to the fruit fly.

[0007] Furthermore, the solid phase microextraction in step (2) includes: 1) Five viable Bactrocera dorsalis were placed in a 2 mL microtube (Eppendorf, Germany), which was immediately frozen with liquid nitrogen and stored in a -80°C ultra-low temperature freezer (MDF-U73V, Sanyo Electric Co. Ltd, Japan). The insect bodies were ground into powder in the microtube and immediately injected into 1.5 mL of HPLC-grade acetonitrile (≥99.9%, Xilong Chemical Co., Ltd., China). The microtube was shaken for 3 min at 1000 rpm using an oscillator (IKA MS 3 basic, IKA, Germany); then centrifuged for 3 min at 25°C and 4000 rpm using a centrifuge (5417R, Eppendorf, Germany); the supernatant (1.3 mL) was taken and transferred to a chromatography bottle (Supelco, Germany); 2) Insert the SPME extraction head (DVB / CAR / pdms, 57348-U, Supelco, USA) into the supernatant sample and extract at 25±1℃ for 1 h. After the extraction, inject the SPME extraction head directly into the GC-MS injection port, desorb at an injection temperature of 270℃ for 15 min, and analyze the sample.

[0008] The method according to claim 1, characterized in that in step (2), an Agilent 8890 gas chromatograph is used, the chromatographic column is an HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm; Agilent) and a 5977B mass detector (MSD); the carrier gas is 99.999% purified helium, with a flow rate of 1 ml / min; the gas chromatography conditions are: the injection port temperature is 270°C; the initial column box temperature is 60°C, maintained for 2 min, then increased to 200°C at a rate of 7°C / min, then increased to 300°C at a rate of 5°C / min, and finally increased to 320°C at a rate of 50°C / min and maintained for 3 min; The mass spectrometry conditions were as follows: the transmission line temperature of the ion source was 230 °C; the transmission line temperature of the MSD was 280 °C; the temperature of the quadrupole was 150 °C; information was collected using the mass spectrometry full scan mode: the mass spectrometry scan range was 30-500 amu, the solvent delay time was 4.5 min, and the total run time was 45.4 min.

[0009] Furthermore, in step (2), the characteristic peaks detected by GC-MS were annotated in the NIST and Wiley Registry metabolome databases according to retention time and m / z, and the OPLS-DA model was used to screen out radiation-related characteristic metabolic markers from the differentially expressed metabolites under different storage conditions.

[0010] The present invention screened out 5 metabolic markers related to the controlled atmosphere irradiation of the oriental fruit fly, which are: 1Docosene, nHexadecanoic acid, Heptadecane -4methyl-, 2Pentadecanone and Tetradecanal.

[0011] In a second aspect, the present invention provides characteristic metabolic markers related to Bactrocera dorsalis obtained according to the method.

[0012] In a third aspect, the present invention provides application of the marker in irradiation detection under different controlled atmosphere storage conditions.

[0013] In a fourth aspect, the present invention provides a method for irradiation detection of Bactrocera dorsalis, the method comprising: S1. Using solid phase microextraction-gas chromatography-mass spectrometry, a PCA model of controlled atmosphere irradiation of Bactrocera dorsalis was established based on the five metabolite markers; S2. Using solid phase microextraction-gas chromatography-mass spectrometry to detect the expression levels of the five metabolite markers in the tested fruit fly to determine whether the tested fruit fly has been irradiated.

[0014] The solid phase microextraction-gas chromatography-mass spectrometry method was as described above.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects.

[0016] The present invention uses solid phase microextraction-gas chromatography-mass spectrometry (GC-MS) for the first time to analyze the metabolites of test insects, thereby studying the metabolic effects of irradiation after gas conditioning treatment on pests. Solid phase microextraction (SPME) is a rapid sample processing technology that can be used to study volatile compounds. Compared with traditional detection methods, solid phase microextraction-gas chromatography-mass spectrometry improves the purity, repeatability and sensitivity of the extract. This method has a good application prospect.

[0017] This method uses a small sample size (5 insects are sufficient), is relatively fast and convenient (no need to ensure that the sample insects are always alive), and can relatively accurately determine whether the citrus fruit fly has been irradiated under different storage conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a PCA score diagram of irradiation of Bactrocera dorsalis in different atmosphere controlled environments in a preferred embodiment of the present invention.

[0020] Figure 2 This is a VIP score diagram of Bactrocera dorsalis irradiated in different atmosphere controlled environments in a preferred embodiment of the present invention.

[0021] Figure 3 This is an analysis diagram of characteristic markers of Bactrocera dorsalis irradiated in different atmosphere controlled environments in a preferred embodiment of the present invention. DETAILED DESCRIPTION The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0023] Example 1 A method for detecting characteristic metabolic markers related to irradiation of Bactrocera dorsalis.

[0024] 1. Materials and methods

[0025] 1.1 Test insect culture The citrus fruit fly was artificially cultured in a guava orchard in the suburbs of Guangzhou at a temperature of 25±1℃, a relative humidity of 65%~75%, and a photoperiod of L:D=14:10, and was used until the third generation.

[0026] 1.2 Controlled atmosphere treatment Eggs within 2 hours were collected and reared to the required age. Healthy larvae were selected as the test insect source and transferred to a 1L controlled atmosphere bag. The bags were filled with 5% nitrogen-balanced oxygen (hypoxia) and 0.5% nitrogen-balanced oxygen (ultra-hypoxia). Air was used as a control. The rearing conditions were left for 12 hours. No less than 100 larvae were used for each treatment and repeated three times.

[0027] 1.3 Irradiation treatment The larvae of Bactrocera dorsalis that had been in controlled atmosphere for 12 hours were kept in the controlled atmosphere and irradiated with 120 Gy together with the controlled atmosphere bag.

[0028] 1.4 SPME extraction Five viable Bactrocera dorsalis were selected and placed in a 2 mL microtube (Eppendorf, Germany). The microtube was immediately frozen with liquid nitrogen and stored in a -80°C ultra-low temperature freezer (MDF-U73V, Sanyo Electric Co. Ltd, Japan). The insect bodies were ground into powder in the microtube and immediately injected into 1.5 mL of HPLC-grade acetonitrile (≥99.9%, Xilong Chemical Co., Ltd., China). The microtube was shaken for 3 min at 1000 rpm using an oscillator (IKA MS 3 basic, IKA, Germany). The microtube was then centrifuged at 25°C and 4000 rpm for 3 min using a centrifuge (5417R, Eppendorf, Germany). The resulting supernatant (1.3 mL) was transferred to a chromatography bottle (Supelco, Germany).

[0029] Insert the SPME extraction head (DVB / CAR / pdms, 57348-U, Supelco, USA) into the supernatant sample. Extract for 1 h at 25±1℃. After extraction, inject the SPME directly into the GC-MS injection port, desorb for 15 min at an injection temperature of 270℃, and analyze the sample.

[0030] 1.5 GC-MS detection An Agilent 8890 gas chromatograph was used, with a HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm; Agilent) and a 5977B mass detector (MSD). The carrier gas was 99.999% purified helium with a flow rate of 1 ml / min. The gas chromatography conditions were: the inlet temperature was 270 °C; the initial temperature of the column box was 60 °C, maintained for 2 min, increased to 200 °C at a rate of 7 °C / min, then increased to 300 °C at a rate of 5 °C / min, and finally increased to 320 °C at a rate of 50 °C / min and maintained for 3 min. The mass spectrometry conditions were: the transmission line temperature of the ion source was 230 °C; the transmission line temperature of the MSD was 280 °C; and the temperature of the quadrupole was 150 °C. Information was collected using full-scan MS mode: the MS scan range was 30–500 amu, the solvent delay time was 4.5 min, and the total run time was 45.4 min.

[0031] 2. Experimental results.

[0032] 2.1 Identification of differentially expressed compounds By processing the GC-MS raw data, samples under different storage conditions were annotated in the NIST and Wiley Registry metabolome databases according to retention time and m / z. A total of 25 metabolites were annotated, including 19 metabolites under hypoxic conditions (Hy), 16 metabolites under ultra-low oxygen conditions (SHY), 23 metabolites under air conditions (Control), 13 metabolites under irradiation treatment (IR), 13 metabolites under hypoxia combined with irradiation treatment (HY+IR), and 10 metabolites under ultra-low oxygen combined with irradiation (SHY+IR), as shown in Table 1.

[0033]

[0034] Note: - indicates not detected.

[0035] 2.2 Screening of radiation characteristic markers.

[0036] like Figure 1 As shown in the figure, the irradiation results of B. dorsalis in different atmospheres were all within the confidence interval, and the different resistances were completely separated without overlap, indicating that the composition and concentration of the molecules contained in these samples were significantly different, and this difference was mainly caused by the difference in compound content. Therefore, it is feasible to use these 25 metabolites as background data to judge the irradiation treatment of B. dorsalis.

[0037] like Figure 2 As shown in the figure, the VIP value (threshold>1) of the OPLS-DA model was used to find differentially expressed metabolites. A total of 5 differential metabolites were screened out after different modified atmosphere irradiation of the orange fruit fly. These 5 compounds can better reflect whether the orange fruit fly has been irradiated. Therefore, it is believed that these compounds have good application prospects as biomarkers for detecting whether the orange fruit fly has been irradiated with modified atmosphere.

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting characteristic metabolic markers related to controlled atmosphere irradiation of Bactrocera dorsalis, characterized in that: The following steps are involved: (1) Taking a certain pest as the research object, using solid phase microextraction and gas chromatography-mass spectrometry to test the pest after irradiation treatment in different atmosphere storage environments, and determine whether the pest has been irradiated based on the test results; (2) Solid phase microextraction-gas chromatography-mass spectrometry was used to detect the metabolites expressed under low oxygen, ultra-low oxygen and normal conditions, and radiation-related characteristic metabolic markers were screened out from the differentially expressed metabolites in different atmospheres.

2. The method according to claim 1, characterized in that The pest is the fruit fly.

3. The method according to claim 1, characterized in that The solid phase microextraction in step (2) includes: (1) Five viable Bactrocera dorsalis were placed in a microtube, which was immediately frozen with liquid nitrogen and stored in a -80°C ultra-low temperature freezer (MDF-U73V, Sanyo Electric Co. Ltd, Japan). The insect bodies were ground into powder in the microtube and immediately injected into 1.5 mL of HPLC-grade acetonitrile (≥99.9%, Xilong Chemical Co., Ltd., China). The microtube was shaken at 1000 rpm using an oscillator for 3 min. The microtube was then centrifuged at 25°C and 4000 rpm in a centrifuge (5417R, Eppendorf, Germany) for 3 min. The supernatant was taken and transferred to a chromatography bottle (Supelco, Germany). (2) Insert the SPME extraction tip into the sample and extract at 25±1℃ for 1 h. After the extraction is completed, inject the SPME extraction tip directly into the GC-MS injection port, desorb at an injection temperature of 270℃ for 15 min, and analyze the sample.

4. The method according to claim 1, characterized in that: In step (2), an Agilent 8890 gas chromatograph was used, the chromatographic column was an HP-5MS capillary column, 30 m × 0.25 mm, 0.25 μm, and a 5977B mass detector; the carrier gas was 99.999% purified helium with a flow rate of 1 ml / min; the gas chromatography conditions were: the inlet temperature was 270°C; the initial column box temperature was 60°C, maintained for 2 min, then increased to 200°C at a rate of 7°C / min, then increased to 300°C at a rate of 5°C / min, and finally increased to 320°C at a rate of 50°C / min and maintained for 3 min; The mass spectrometry conditions were as follows: the transmission line temperature of the ion source was 230 °C; the transmission line temperature of the MSD was 280 °C; the temperature of the quadrupole was 150 °C; information was collected using the mass spectrometry full scan mode: the mass spectrometry scan range was 30-500 amu, the solvent delay time was 4.5 min, and the total run time was 45.4 min.

5. The method according to claim 4, characterized in that In step (2), the characteristic peaks detected by GC-MS were annotated in the NIST and Wiley Registry metabolome databases according to retention time and m / z, and the OPLS-DA model was used to screen out radiation-related characteristic metabolic markers from the differentially expressed metabolites in different modified atmosphere environments.

6. The method according to any one of claims 2 to 5, characterized in that: A total of five metabolic markers related to irradiation of Bactrocera dorsalis were screened out, namely: 1Docosene, nHexadecanoic acid, Heptadecane -4methyl-, 2Pentadecanone and Tetradecanal.

7. Pest irradiation-related characteristic metabolic markers obtained according to the method of any one of claims 1 to 6.

8. Use of the marker according to claim 7 in pest irradiation detection.

9. A method for detecting the citrus fruit fly by irradiation, characterized in that: The method comprises: S1. Using solid phase microextraction-gas chromatography-mass spectrometry to identify whether the fruit fly has been irradiated under different storage and transportation conditions based on the five metabolite markers described in claim 6; S2. Using solid phase microextraction-gas chromatography-mass spectrometry to detect the expression levels of the five metabolite markers in the tested fruit fly, and substituting them into the PCA model to determine whether the tested fruit fly has been irradiated.

10. The method according to claim 9, characterized in that The solid phase microextraction-gas chromatography-mass spectrometry method is the same as that described in any one of claims 1-5.

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