CN109828019A

CN109828019A - The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen

Info

Publication number: CN109828019A
Application number: CN201910129821.8A
Authority: CN
Inventors: 罗丽萍; 郝影宾; 陈焕文; 薛阿辉; 刘永泉; 崔萌; 郭夏丽; 吴栋
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2019-02-21
Filing date: 2019-02-21
Publication date: 2019-05-31

Abstract

The present invention provides a kind of method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen, by electron spray extraction ionization mass spectrometry technology measurement Citrus leaf metabolin variation, in conjunction with principal component analytical method, before the common detection methods such as PCR are difficult to effectively detect susceptible nursery stock, the citrus nursery stock of infection early stage in yellow twig incubation period is identified.In the lab, it detects sample 30 times, detection effect is up to 98%；The field trial stage is detected sample 100 times, and detection effect effectively can quickly detect Citrus Huanglongbing pathogen up to 90%.

Description

The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen

Technical field

The present invention is a kind of rapid detection method of Citrus Huanglongbing pathogen, belongs to plant disease detection method field.

Background technique

Citrus Huanglongbing pathogen is the great destructive disease of one of Orange Producing, parasitizes plant phloem group by one kind The bacterium knitted causes, including Asia Candidatus liberibacter, African Candidatus liberibacter and America Candidatus liberibacter.It is entire after plant is susceptible Tree vigo(u)r weakens, slow growth, and blade is in mottled yellow, and fruit color is uneven, and " red nose fruit " symptom is presented, and taste of fruit is thin out, Treelet is withered generally in 2-3, and adult trees are withered in 5-8, seriously reduces the economic benefit of citrus.It is so far Only, the effective agent and resistant variety for the disease are had no, therefore the diagnosis early of disease is the base of Citrus Huanglongbing pathogen prevention and control Plinth is also crucial.The method for really being able to make a definite diagnosis is laboratory diagnosis, needs sections observation by microscopy, there are larger Missing inspection may；Serology, nucleic acid hybridization and the methods of PCR can the detection when bacteria concentration reaches a certain level, in plant sense Disease preclinical early stage fails to detect.And this method can be in the susceptible preclinical early stage detection of plant, maximum can The susceptible nursery stock of judgement of energy, avoids more huge economic loss.

Summary of the invention

Sample picks up from Sensing Technique in Xingguo County, Jiangxi Province navel orange garden；CK is healthy leaves；T0 is incubation period blade (after grafting yellow twig bud 10th week)；T1 is infection Citrus Huanglongbing pathogen blade.

The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen, comprising the following steps:

(1) fresh Citrus leaf is acquired, 0.2000g sample is accurately weighed, in pulverized under liquid nitrogen at powder, is mentioned with DNA of plants Kit is taken to extract DNA, PCR reaction system total volume is 20 μ l；(2) PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94℃ It is denaturalized 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 40s, is recycled 35 times；72 DEG C of extension 10min；

(3) agarose gel electrophoresis program: using 1% agarose glue, and electrophoretic buffer is 1 × TBE buffer, 120V constant pressure electrophoresis 30min, using DNALadder as DNA molecular amount standard；Under gel imaging system, observation, preservation of taking pictures；

(4) 70% methanol 2mL is added at powder in precise navel orange leaf fresh weight 0.20g, pulverized under liquid nitrogen, and ultrasonic wave added mentions 35min is taken, 10000 × g is centrifuged 10min at 4 DEG C, takes supernatant, 0.22 μm of membrane filtration is to be measured；

(5) linear ion hydrazine mass spectrum (LTQ-MS) is negative ion mode measurement, the source ESI, scanning of the mass spectrum range m/z 50- 1000；150 DEG C of ion transfer tube temperature；ESI solvent is methanol；Atomization gas is nitrogen, pressure 1.2MPa；ESI solvent and sample Product flow velocity is respectively 4,5 μ L/min；The distance between two atomizing nozzles 1mm, 60 ° of angle, apart from mass spectrum mouth 5mm, angle is 150 °； Accurate mass and NIST that (6) the electron spray compound characterization that detects of extraction ionization mass spectrometry is obtained by control experiment, Tandem mass spectrum information and standard items spectrogram determine in Metlin and Pub Chem database, carry out principal component using Matlab software Analysis.

The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen: the PCR reaction system group in step (1) Become: 16S rDNA special primer each 1 μ l of Las606/LSS of 2 × Mix, 10 μ l, template DNA 1 μ g, CLas, distilled water are supplied 20μl。

The method that the electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen: in step (5) when measurement Experiment condition are as follows: extractant is methanol, electron spray voltage 3.5kV, and parent ion isolation window width is set as 1.0Da, when collision Between be 30ms, collision energy 15%-35%, other parameters are by LTQ-MS software systems Automatic Optimal.

Beneficial effects of the present invention

The present invention is by electron spray extraction ionization mass spectrometry technology measurement Citrus leaf metabolin variation, in conjunction with principal component analysis Method identifies infection yellow twig incubation period early stage rank before the common detection methods such as PCR are difficult to effectively detect susceptible nursery stock The citrus nursery stock of section.

Detailed description of the invention

Fig. 1 is that electron spray extracts ionization mass spectrometry schematic diagram and device pictorial diagram.

Fig. 2 is the PCR result figure of healthy leaf (CK), incubation period leaf (T0) and susceptible leaf (T1).

Fig. 3 is the principal component analysis figure of healthy leaf and incubation period leaf, and wherein CK is healthy leaf, and T0 is incubation period leaf.

Specific embodiment

Embodiment 1

The method that the electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen: in step (5) when measurement Experiment condition are as follows: extractant is methanol, electron spray voltage 3.5kV, and parent ion isolation window width is set as 1.0Da, when collision Between be 30ms, collision energy 25%, other parameters are by LTQ-MS software systems Automatic Optimal.

By laboratory and field trial show that Citrus Huanglongbing pathogen can not be detected in normal PCR analysis method preclinical Disease plant, and electron spray of the present invention extracts ionization mass spectrometry technology, can quickly detect plant in conjunction with principal component analytical method Susceptible preclinical early stage plant, the susceptible nursery stock of the judgement of maximum possible avoid more huge economic loss.In laboratory In, it detects sample 30 times, detection effect is up to 98%；It in the field trial stage, detects sample 100 times, detection effect is up to 90%.

Claims

1. the method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen, which comprises the following steps:

(1) fresh Citrus leaf is acquired, 0.2000g sample is accurately weighed, in pulverized under liquid nitrogen at powder, is extracted and is tried with DNA of plants Agent box extracts DNA, and PCR reaction system total volume is 20 μ l；

(2) PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 40s, circulation 35 times；72 DEG C of extension 10min；

(3) agarose gel electrophoresis program: using 1% agarose glue, and electrophoretic buffer is 1 × TBE buffer, and 120V is permanent Piezoelectricity swimming 30min, using DNA Ladder as DNA molecular amount standard；Under gel imaging system, observation, preservation of taking pictures；

(4) 70% methanol 2mL, ultrasound assisted extraction is added at powder in precise navel orange leaf fresh weight 0.20g, pulverized under liquid nitrogen 35min, 10000 × g is centrifuged 10min at 4 DEG C, takes supernatant, 0.22 μm of membrane filtration is to be measured；

(5) use linear ion hydrazine mass spectrum (LTQ-MS) for negative ion mode measurement, the source ESI, scanning of the mass spectrum range m/z 50- 1000；150 DEG C of ion transfer tube temperature；ESI solvent is methanol；Atomization gas is nitrogen, pressure 1.2MPa；ESI solvent and sample Product flow velocity is respectively 4,5 μ L/min；The distance between two atomizing nozzles 1mm, 60 ° of angle, apart from mass spectrum mouth 5mm, angle is 150 °；

(6) accurate mass that the electron spray compound characterization that detects of extraction ionization mass spectrometry is obtained by control experiment with Tandem mass spectrum information and standard items spectrogram determine in NIST, Metlin and Pub Chem database, are carried out using Matlab software Principal component analysis.

2. the method that electron spray extraction ionization mass spectrometry according to claim 1 quickly detects Citrus Huanglongbing pathogen, feature It is: the PCR reaction system composition in step (1) are as follows: the 16S rDNA of 2 × Mix10 μ l, template DNA 1 μ g, CLas specifically draw Each 1 μ l of object Las606/LSS, distilled water supply 20 μ l.

3. the method that electron spray extraction ionization mass spectrometry according to claim 1 quickly detects Citrus Huanglongbing pathogen, feature It is: the experiment condition in step (5) when measurement are as follows: extractant is methanol, electron spray voltage 3.5kV, parent ion isolation window Width is set as 1.0Da, collision time 30ms, collision energy 15%-35%, and other parameters are automatic by LTQ-MS software systems Optimization.