CN110927318A - Method for rapidly identifying thrips by GC-MS - Google Patents
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Abstract
The invention relates to a GC-MS rapid identification method for thrips, which comprises the following steps: placing the adult thrips/larva in front of an insect epidermal hydrocarbon heat adsorption sample injection device, injecting a normal alkane C21-C40 standard sample to obtain each alkane chromatographic peak in the normal alkane standard sample, wherein the normal alkane retention time is used as a standard; calculating the Kovat retention index Kov { hacek over (a) } ts index, KI of epidermal hydrocarbons contained in the sample; and so on. The method is quick, effective, simple and convenient, and can provide technical support for quick quarantine of thrips, particularly adult/larva quarantine.
Description
Technical Field
The invention belongs to the technical field of thrips identification, and particularly belongs to the technical field of a rapid thrips GC-MS identification method.
Background
Thrips (Thysanoptera: Thripidae) is a large group of insects, of which about 6000 species are known in the world and 566 species exist in china. Several hundreds of thrips worldwide are important pests of vegetables, fruits and ornamental plants, which have high reproductive capacity and drug resistance. Some of these are the major pests on vegetables and ornamentals, others, such as thrips occidentalis, Frankliniella occidentalis (Pergande), are among the major invasive pests. Besides morphological classification, molecular methods are mainly used for thrips identification.
Recent studies have shown that epidermal hydrocarbons (CHCs) of insects are used for chemical classification of species and subspecies. Traditionally, solvent liquid samples are a common method of extracting and analyzing insect CHCs. However, there is still a technical problem in the identification of thrips larvae, and even a taxonomist cannot accurately classify the larvae. In most cases, the adult insects are raised and then subjected to species identification according to the morphological characteristics of the adult insects, or molecular biology techniques are required. In addition, in the quarantine inspection of invasive pests, when the number of insects is small, it is difficult to satisfy molecular identification or to obtain sufficient hydrocarbon amount by solvent extraction for identification. In recent years, the solid sample injection technology can quickly and simply extract and analyze CHC of single-head insects, even insect body attachments or insect fragments, and is very suitable for CHC analysis of micro insects such as thrips, aphids and the like. The direct sampling technology for extracting the hydrocarbon on the insect epidermis provides a new simple and quick method for identifying the insects, only one insect sample, even the stump of the insect is needed, and solvent dissolution is not needed.
Frankliniella occidentalis is native to North America and is one of the important invading pests on many vegetable and ornamental crops. Since the discovery in Beijing of China in 2003, the plants are distributed in the southeast, southwest and east China. To date, such insects have been detected only in cut flowers shipped from Yunnan, but not in vegetables and ornamentals from Guangdong province. Frankliniella intnsa, Thrips hawaiiensis (Morgan), Thrips palmi Thripspmi, Thrips sinensis Prinsensins Primer and Thrips banyanensis Gynaikothrips ficum (Marchal) are some of the Thrips common on vegetables and ornamental plants in Guangdong province. The adult thrips of the genus thrips and the genus thrips are not easily identified in morphology, and the larvae are more difficult to identify. Therefore, a rapid and simple thrips identification method is very important for quarantine monitoring of frankliniella occidentalis.
Disclosure of Invention
The invention provides a method for rapidly identifying thrips by GC-MS (gas chromatography-Mass spectrometer), which is capable of clearly recognizing the defects of the problems. The method is quick, effective, simple and convenient, and can provide technical support for quick quarantine of thrips, particularly adult/larva quarantine.
The invention is realized by adopting the following technical scheme.
A method for GC-MS rapid identification of thrips comprises the following steps: placing the adult thrips/larva in an insect epidermis hydrocarbon heat adsorption sample injection device, injecting a normal alkane C21-C40 standard substance to obtain each alkane chromatographic peak in the normal alkane standard substance, wherein the normal alkane retention time is used as a standard.
Further, the method of the invention comprises the following steps: calculating the Kovat retention index Kov { hacek over (a) } ts index, KI:
KI values for the epidermal hydrocarbon components of thrips test samples were calculated according to the following formula: KI 100n +100 × [ tR (x) -tR (n)) ]/[ tR (n +1) -tR (n)) ]
"n" and "n + 1" represent the number of carbon atoms of the normal alkane in front of and behind a certain chromatographic peak of a sample to be identified, and "tR (n)", and "tR (n + 1)" are retention times of adjacent normal alkane chromatographic peaks in front of and behind a certain chromatographic peak of a sample to be detected; "tR (x)" represents the retention time of a certain chromatographic peak of a sample to be measured.
Further, the method of the invention comprises the following steps: the method comprises the steps of identifying each component obtained by gas chromatography separation through a mass spectrometer by using a gas chromatography-mass spectrometry combined technology, firstly deducing the molecular weight of each component according to fragment peak and molecular ion peak information obtained by a mass spectrogram of each component, then carrying out primary analysis according to a general rule of molecular fragmentation and a mass spectrometry analysis program, and then determining the structural formula of each component by comparing with EPA/NzHMas and spectralData standard spectrograms.
Further, the method of the invention comprises the following steps: and analyzing the thrips epidermal hydrocarbons by using a gas chromatograph, and respectively integrating the area of each component peak according to the obtained total ion current chromatogram of each test insect to obtain the area of each corresponding component peak.
Further, the method of the invention comprises the following steps: and (3) calculating the relative content of the components according to the peak area of each component in proportion, and taking the relative content as a quantitative index of each component in the test insect epidermal hydrocarbon mixture.
Further, the method of the invention comprises the following steps: and (3) performing multivariate pattern recognition analysis on peak area data of each converted epidermal hydrocarbon of thrips by adopting a Principal Component Analysis (PCA).
Further, the method of the invention comprises the following steps: the original variables are converted into a new set of principal component variables using orthogonal linear transformation, and then the variables with significant discriminative power are screened out using Discriminant Analysis (DA).
Further, the method of the invention comprises the following steps: the standard set-up was preceded by a sample preparation step: freezing multiple head imagoes/nymphs/larvae of different species of thrips in a refrigerator for 0.5h for later use, and respectively placing 1 head imagoes/larvae in an insect epidermis hydrocarbon heat adsorption sample injection device for later use; GC-MS spectrum analysis was performed on adult/larval thrips.
Further, the method of the invention comprises the following steps: the sample injection device with the test insects is placed at the temperature of 280 ℃ of a sample injection port and is analyzed for 5min in a non-flow-dividing sample injection mode.
Further, the method of the invention comprises the following steps: the GC-MS spectrum analysis parameters are as follows: the chromatographic column is HP-5MS S, 5% dimethyl biphenyl-95% dimethyl polysiloxane, 30m × 0.2mm, 0.25-mum membrane; the procedure of the furnace temperature was: maintaining the initial temperature at 80 deg.C for 1min, increasing the temperature to 200 deg.C at 5 deg.C/min, maintaining for 2min, increasing the temperature to 280 deg.C at 10 deg.C/min, and maintaining for 10 min; nitrogen was used as the carrier gas and the column pressure was 9.32 psi.
The method has the beneficial effects that according to species specificity of the epidermal hydrocarbons of insects, the epidermal hydrocarbons of the Frankliniella occidentalis and several common adult/nymph/larva of the Frankliniella occidentalis with similar forms are researched by utilizing a technology of combining direct solid sample injection and gas chromatography-mass spectrometry (GC-MS), and the GC-MS rapid identification method of the Frankliniella occidentalis is provided. The method is quick and effective, and can provide necessary technical support for quick customs clearance of customs quarantine.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a total ion flow diagram of epidermal carbon compounds of six types of adult thrips. (A, B: Holland and Beijing population of Frankliniella occidentalis, C: Frankliniella occidentalis, D: Frankliniella palmipennis, E: Frankliniella flaviperidis, F: Frankliniella sinensis, G: Frankliniella banorhii);
FIG. 2 is a graph of the discriminant analysis of the relative hydrocarbon content of the epidermis of six species of adult thrips.
FIG. 3 is a total ion flow graph of epidermal carbon compounds of three thrips nymphs. (A, B: Holland and Beijing population of Frankliniella occidentalis, C: thrips palmi, D: Frankliniella flavipes).
FIG. 4 is a graph of the discriminant analysis of the relative hydrocarbon content of the epidermal of three thrips nymphs.
Detailed Description
The following are specific embodiments of the present invention, which are provided for explaining the technical contents and actual effects of the present invention and do not provide any limitation to the protection of the present invention.
Sample preparation
Freezing multiple head imagoes and nymphs of different species of thrips in a refrigerator for 0.5h for later use, and respectively placing 1 head imagoes or nymphs in an insect epidermis hydrocarbon heat adsorption sampling device for later use.
GC-MS spectrum analysis is carried out on the thrips imago nymphs.
The GC-MS analysis instrument is a gas chromatography-mass spectrometry combined instrument 7890A-5975C manufactured by Agilent.
The sample injection device with the test insects is placed at the temperature of 280 ℃ of a sample injection port and is analyzed for 5min in a non-flow-dividing sample injection mode.
The column was HP-5MS S (5% dimethylbiphenyl-95% dimethylpolysiloxane, 30 m.times.0.2 mm, 0.25-. mu.m membrane).
The procedure of the furnace temperature was: the initial temperature is maintained at 80 deg.C for 1min, raised to 200 deg.C at 5 deg.C/min, maintained for 2min, and then raised to 280 deg.C at 10 deg.C/min, and maintained for 10 min.
The carrier gas was high purity nitrogen and the column pressure was 9.32 psi.
Data analysis
80ng normal paraffin C21-C40 standard (Sigma, USA) GC-MS was first introduced before sample injection. And (3) obtaining each alkane chromatographic peak in the normal alkane standard product, and calculating the Kovat retention index (Kov { hacek over (ts) } index, KI) of epidermal hydrocarbons contained in the sample by taking the retention time of the normal alkanes as a standard.
KI values for the epidermal hydrocarbon components of thrips test samples were calculated according to the following formula: KI 100n +100 x [ t ]R(x)－tR(n)]/[tR(n+1)－tR(n)]
"n" and "n + 1" represent the number of carbon atoms of the normal alkane preceding and following a certain chromatographic peak of the sample to be identified, "t" isR(n)"and" tR(n+1)"is the retention time of the adjacent normal alkane chromatographic peak before and after a certain chromatographic peak of a sample to be detected; "t" sR(x)"indicates the retention time of a certain chromatographic peak of the sample to be tested.
The method comprises the steps of identifying each component obtained by gas chromatography separation through a mass spectrometer by using a gas chromatography-mass spectrometry combined technology, firstly deducing the molecular weight of each component according to fragment peak and molecular ion peak information obtained by a mass spectrogram of each component, then carrying out primary analysis according to a general rule of molecular fragmentation and a mass spectrometry analysis program, and then determining the structural formula of each component by comparing with EPA/NzHMas and spectralData standard spectrograms.
Analyzing the thrips epidermal hydrocarbons by using a gas chromatograph, and respectively integrating the area of each component peak according to the obtained total ion current chromatogram of each test insect to obtain a group of corresponding area of each component peak (when the experimental conditions are constant, the peak area or peak height is in direct proportion to the content of the components).
And (3) calculating the relative content of the components according to the peak area of each component in proportion, and taking the relative content as a quantitative index of each component in the test insect epidermal hydrocarbon mixture.
And (3) performing multivariate pattern recognition analysis on peak area data of each converted epidermal hydrocarbon of thrips by adopting a Principal Component Analysis (PCA).
The original variables are converted into a new set of principal component variables using orthogonal linear transformation, and then the variables with significant discriminative power are screened out using Discriminant Analysis (DA).
Statistical analysis used Statistica 10.0(Statsoft, Inc.).
Test one: method for analyzing differences of epidermal hydrocarbons of different geographical populations of Frankliniella occidentalis
TABLE 1 Frankliniella occidentalis different geographical population epidermal hydrocarbons
Respectively carrying out GC-MS analysis on epidermal hydrocarbons of Beijing population of Frankliniella occidentalis, Netherlands population adults and nymphs, and detecting that 9 hydrocarbons exist on the epidermis of the Beijing population adults, 8 hydrocarbons exist on the nymphs, 10 hydrocarbons exist on the epidermis of the Netherlands population adults, and 9 hydrocarbons exist on the nymphs, which are straight-chain or branched-chain saturated alkanes (Table 1). Among them, 9, 14-dimethyltriacontane is a compound unique to nymphs in the Netherlands population but not in the Beijing population. The similarity of GC-MS analysis maps of imagoes among different geographical populations of Frankliniella occidentalis is as high as 90.00%.
And (2) test II: epidermal hydrocarbon species and analysis of six common thrips adults
TABLE 2 epidermal hydrocarbon species of six common adult thrips
GC-MS analysis of epidermal hydrocarbons of 6 common thrips adults, such as Frankliniella occidentalis, Frankliniella palmata, Frankliniella banorum and Frankliniella tersimply, is carried out to obtain an epidermal carbon compound total ion flow diagram (figure 1) of 6 thrips adults, to obtain 15 hydrocarbons, namely n-pentacosane, 9-methyl pentacosane, 3-methyl pentacosane, n-hexacosane, n-heptacosane, 9, 11, 13-trimethyl heptacosane, 7-methyl heptacosane, 5-methyl heptacosane, 3-methyl heptacosane, n-nonacosane, 11, 13, 15-trimethyl nonacosane, 7-methyl nonacosane, 9, 14-dimethyl triacontane, 5-methyl triacontane, 3-methyl triacontane, which are straight-chain or branched saturated alkanes (Table 2).
The peak areas of 15 hydrocarbons of 6 kinds of thrips adults were logarithmically transformed, and then the main components were performed. Four main components are obtained. The total variance of these four principal components was 34.2%, 28.6%, 15.9%, and 8.5%, respectively, accounting for 87.23% of the original variance.
The factor scores of the 4 principal components are subjected to discriminant analysis, the analysis shows that 6 thrips epidermal hydrocarbons have significant differences (Walks' lambda <0.0001, approximate F ═ 244.3553, p <0.001), and 4 characteristic roots are obtained in the discriminant analysis, and the 4 characteristic roots are shown to have extremely significant statistical significance through the Chi-square test, wherein root 1 can explain 48.4% of the variation, root 2 can explain 31.3% of the total variation, and 4 characteristic roots explain 100% of the total variation. The interactive verification method carries out the back judgment on each kind of thrips imagoes, and the accuracy rate is 100 percent. The dot-plot of discriminant analysis showed that there was significant separation between adults of Frankliniella occidentalis, Frankliniella palmipennis, Frankliniella banyana, and Frankliniella terniformis (FIG. 2). The statistical results show that: root 1 can clearly distinguish ficus microcarpa, Chinese simple tube thrips and flower thrips (p < 0.001). Root 2 clearly distinguished thrips palmi, thrips occidentalis and thrips palmi (p < 0.001).
And (3) test III: epidermal hydrocarbon species and analysis of three common thrips larvae
TABLE 3 epidermal hydrocarbon species of three common adult thrips
The GC-MS analysis of epidermal hydrocarbons of 3 common thrips larvae of Frankliniella occidentalis, thrips palmi and thrips flavicans is carried out to obtain a total ion flow diagram (figure 3) of epidermal hydrocarbons of 3 thrips larvae, and 11 hydrocarbons are respectively n-pentacosane, 9-methyl pentacosane, 3-methyl pentacosane, n-hexacosane, n-heptacosane, 9, 11, 13-trimethyl heptacosane, 9-methyl heptacosane, 5-methyl heptacosane, 3-methyl heptacosane, n-nonacosane, 11, 13 and 15-trimethyl nonacosane, and are straight-chain or branched-chain saturated alkanes (Table 3).
The peak areas of 11 hydrocarbons of 3 thrips larvae were logarithmically transformed and then subjected to principal component. Two main components are obtained. The total variance of these two principal components was 47.2% and 42.5%, respectively, accounting for 89.61% of the original variance.
The above-mentioned factor scores of 2 principal components were subjected to discriminant analysis, which revealed that 3 thrips epidermal hydrocarbons had significant differences (Walks' λ <0.0001, approximate F ═ 386.9106, p <0.001), and 2 characteristic roots were obtained in discriminant analysis, all of which had very significant statistical significance by chi-square test, wherein root 1 could explain 77.4% of the variation, root 2 could explain 22.5% of the total variation, and 2 characteristic roots explain 99.9% of the total variation. The interactive verification method carries out the back judgment on each kind of thrips imagoes, and the accuracy rate is 100 percent. The dot plots from the discriminant analysis showed significant separation between thrips occidentalis, thrips palmi, and thrips palmi larvae (fig. 4). The statistical results show that: root 1 can clearly distinguish between Frankliniella occidentalis and Frankliniella flavipes (p < 0.001). Root 2 clearly distinguished thrips palmi from thrips flavipes (p < 0.001).
The above description is only a part of specific embodiments of the present invention (since the technical solution of the present invention includes parameters, the embodiments are not exhaustive, and the protection scope of the present invention is subject to the parameters of the present invention and other technical points), and the specific contents or common sense known in the solutions are not described herein too much. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. A method for rapidly identifying thrips by GC-MS is characterized by comprising the following steps: placing the adult thrips into an insect epidermis hydrocarbon heat adsorption sample injection device, injecting a normal alkane C21-C40 standard sample to obtain each alkane chromatographic peak in the normal alkane standard sample, and using the retention time of the normal alkane as a standard.
2. The method for GC-MS rapid identification of thrips according to claim 1, which comprises the following steps: calculating the Kovat retention index Kov { hacek over (a) } ts index, KI:
KI values for the epidermal hydrocarbon components of thrips test samples were calculated according to the following formula: KI 100n +100 × [ tR (x) -tR (n)) ]/[ tR (n +1) -tR (n)) ]
"n" and "n + 1" represent the number of carbon atoms of the normal alkane in front of and behind a certain chromatographic peak of a sample to be identified, and "tR (n)", and "tR (n + 1)" are retention times of adjacent normal alkane chromatographic peaks in front of and behind a certain chromatographic peak of a sample to be detected; "tR (x)" represents the retention time of a certain chromatographic peak of a sample to be measured.
3. The method for GC-MS rapid identification of thrips according to claim 1, which comprises the following steps: the method comprises the steps of identifying each component obtained by gas chromatography separation through a mass spectrometer by using a gas chromatography-mass spectrometry combined technology, firstly deducing the molecular weight of each component according to fragment peak and molecular ion peak information obtained by a mass spectrogram of each component, then carrying out primary analysis according to a general rule of molecular fragmentation and a mass spectrometry analysis program, and then determining the structural formula of each component by comparing with EPA/NzHMas and spectralData standard spectrograms.
4. The method for GC-MS rapid identification of thrips according to claim 3, which comprises the following steps: and analyzing the thrips epidermal hydrocarbons by using a gas chromatograph, and respectively integrating the area of each component peak according to the obtained total ion current chromatogram of each test insect to obtain the area of each corresponding component peak.
5. The method for GC-MS rapid identification of thrips according to claim 4, which comprises the following steps: and (3) calculating the relative content of the components according to the peak area of each component in proportion, and taking the relative content as a quantitative index of each component in the test insect epidermal hydrocarbon mixture.
6. The GC-MS rapid identification method for thrips according to claim 1, characterized in that the method comprises the following steps: and (3) performing multivariate pattern recognition analysis on peak area data of each converted epidermal hydrocarbon of the thrips by adopting a principal component analysis method.
7. The GC-MS rapid identification method for thrips according to claim 6, characterized in that the method comprises the following steps: the original variables are converted into a group of new principal component variables by utilizing orthogonal linear transformation, and then the variables with remarkable discriminant ability are screened out by discriminant analysis.
8. The GC-MS rapid identification method for thrips according to claim 1, characterized in that the method comprises the following steps: the standard set-up was preceded by a sample preparation step: freezing multiple head imagoes/larvae of different species of thrips in a refrigerator for 0.5h for later use, and respectively placing 1 head imagoes/larvae in an insect epidermis hydrocarbon heat adsorption sample injection device for later use; GC-MS spectrum analysis was performed on adult/larval thrips.
9. The GC-MS rapid identification method for thrips according to claim 8, characterized in that the method comprises the following steps: the sample injection device with the test insects is placed at the temperature of 280 ℃ of a sample injection port and is analyzed for 5min in a non-flow-dividing sample injection mode.
10. The GC-MS rapid identification method for thrips according to claim 8, characterized in that the method comprises the following steps: the GC-MS spectrum analysis parameters are as follows: the chromatographic column is HP-5MS S, 5% dimethyl biphenyl-95% dimethyl polysiloxane, 30m × 0.2mm, 0.25-mum membrane; the procedure of the furnace temperature was: maintaining the initial temperature at 80 deg.C for 1min, increasing the temperature to 200 deg.C at 5 deg.C/min, maintaining for 2min, increasing the temperature to 280 deg.C at 10 deg.C/min, and maintaining for 10 min; nitrogen was used as the carrier gas and the column pressure was 9.32 psi.
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