CN107312869B - Kit for detecting silkworm microsporidian by PCR-ELISA method and detection method thereof - Google Patents

Kit for detecting silkworm microsporidian by PCR-ELISA method and detection method thereof Download PDF

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CN107312869B
CN107312869B CN201710774384.6A CN201710774384A CN107312869B CN 107312869 B CN107312869 B CN 107312869B CN 201710774384 A CN201710774384 A CN 201710774384A CN 107312869 B CN107312869 B CN 107312869B
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潘国庆
王锐
周泽扬
宋跃
何章帅
刘璐璐
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Xunjie Chongqing Life Technology Co ltd
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Abstract

The invention relates to a kit for detecting nosema bombycis by a PCR-ELISA method and a detection method thereof, wherein the kit comprises a primer pair for detecting nosema bombycis marked by a nucleic acid molecular marker and an ELISA reagent for detecting the nucleic acid molecular marker; through amplifying appropriate gene segments by using the labeled specific primer pair and then carrying out ELISA on the amplified product, the screened primer has species specificity and high sensitivity, can realize high-throughput detection of multiple samples, and has important significance for detecting the nosema bombycis in finished eggs of silkworms.

Description

Kit for detecting silkworm microsporidian by PCR-ELISA method and detection method thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a kit for detecting nosema bombycis by a PCR-ELISA method, and a detection method.
Background
Nosema bombycis (Nosema bombycis) is a common pathogenic fungus of silkworms, and the Nosema bombycis caused by the Nosema bombycis is a destructive disease in the mulberry industry, so that the Nosema bombycis is always listed as a legal silkworm production quarantine object by various silkworm breeding countries and regions. In order to effectively control the nosema bombycis in the silkworm industry to prevent the silkworm from disastrous death, how to complete the detection of the nosema bombycis in the egg-finished stage becomes one of the important works in the scientific research of the silkworm industry.
In the 60's of 19 th century, Pasteur (Louis Pasteur) discovered and utilized the infection characteristics of silkworm corpuscle disease embryoid, established a prevention and control technology which can eliminate the eggs laid by the infected female moths by checking the infection condition of the corpuscle disease of the female moths and ensure that the eggs of offspring are disease-free, and became the key technology for preventing and controlling the silkworm corpuscle disease. With the shift of the scale, mode and technology of silkworm breeding or silkworm breeding production, the technology is greatly improved, and the improvement is mainly shown as follows: the single moth full detection is developed into group female moth sampling detection, so that the detection efficiency is higher, the first generation hybrid adopts a judgment standard which allows a certain disease moth rate, so that the silkworm egg production under the detection system is more economical, the detection result is more accurate due to the improvement of the detection related facility equipment conditions, and the like. The mother moth microscopic examination method belongs to indirect examination, and uses a microscope to identify the existence of microsporidian in the body of the mother moth according to the characteristics of microsporidian, such as form, refractivity and the like, and further judges whether microsporidian exists in the silkworm eggs. The method is simple and convenient to operate, results are intuitive and easy to understand, but because the requirement on detection personnel is high, the preparation of a female moth sample is complex, meanwhile, nosema bombycis and fungal spores, host tissue fragments and the like are easy to be confused in microscopic examination work, and the speed and the accuracy of the detection work are reduced, so that the establishment of the method for directly detecting the nosema bombycis in finished eggs becomes the key point of scientific research in the silkworm industry.
With the progress of technology and the update of instruments and equipment, more and more novel detection methods are applied to the detection of nosema bombycis, most of the detection methods are based on nucleic acid detection, such as a PCR method, a LAMP method, a fluorescence quantitative PCR and the like, the detection methods have higher sensitivity and accuracy compared with the traditional female moth microscopic examination, but some methods have higher requirements on operation of inspectors, need to be trained professionally, have higher dependence on instruments and equipment and higher cost, and are still in the laboratory research stage at present. Therefore, the development of a method for directly detecting the microsporidian in the finished eggs with high efficiency, convenience, sensitivity, accuracy, lower cost and high flux has great significance for the prevention and control of the nosema bombycis.
In 1997, Niemeyer et al created a PCR-ELISA technique. The upstream and downstream primers are marked, a PCR product is incubated with an enzyme label plate of pre-coated streptavidin by means of a biotin-streptavidin system, an enzyme-labeled antibody is added for reaction, and finally, a substrate is added for color development to determine the OD value, so that the existence of a certain specific gene is detected by the method. The method makes up the deficiency of nucleic acid detection, and experiments at home and abroad show that the sensitivity of PCR-ELISA is 10-100 times higher than that of agarose electrophoresis; in addition, the reading result of the microplate reader is objective, and the subjective interference is small; the DNA extraction, the PCR amplification, the incubation and the color development of the microplate and the judgment result can be finished in one working day at the fastest speed, and the used time is short; multiple samples can be detected on one microporous plate simultaneously, so that high-throughput rapid detection is realized; the reagent is common buffer solution, the preparation is simple, and the used instruments are as follows: the microplate reader, the PCR instrument and the incubator are possessed by most laboratories, and are favorable for basic popularization and application.
In conclusion, in the detection method, the special marker is added in the nucleic acid amplification process, the PCR product is used as the antigen to carry out ELISA detection, the sample can be subjected to high-throughput detection, and the result interpretation is objective.
Disclosure of Invention
In view of the above, one of the objectives of the present invention is to provide a kit for detecting nosema bombycis by PCR-ELISA; the invention also aims to provide a method for detecting the nosema bombycis by a PCR-ELISA method.
In order to achieve the purpose, the invention provides the following technical scheme:
the kit for detecting the nosema bombycis by the PCR-ELISA method comprises a primer pair for detecting the nosema bombycis marked by a nucleic acid molecular marker and an ELISA reagent for detecting the nucleic acid molecular marker; the sequences of the primer pairs are shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.75 and SEQ ID NO.76 or SEQ ID NO.77 and SEQ ID NO. 78.
Preferably, the sequences of the primer pair are shown as SEQ ID NO.49 and SEQ ID NO. 50.
Preferably, the ELISA reagent for detecting the nucleic acid molecular marker comprises the following components in concentration: 1 mu g/mL streptavidin, PBS solution containing 2% fetal calf serum by volume fraction, enzyme-labeled DIG antibody, PBST solution, and H with 30% by volume fraction2O2500mM oxalic acid.
Preferably, the nucleic acid molecular markers are biotin and digoxin.
More preferably, the kit further comprises a sample processing reagent, wherein the sample processing reagent comprises 10% of KOH by mass, STP lysate, glass beads with the diameter of 425 and 600 μm, 20mg/mL proteinase K, 10% of SDS by mass, phenol: chloroform: mixed solution of isoamylol with volume ratio of 25:24:1 and absolute ethyl alcohol; the concentration of each component of the STP lysate is as follows: 1% mass fraction SDS, 1% mass fraction Triton X-100 and 1% mass fraction Nonidet P40.
2. The PCR-ELISA method for detecting silkworm microsporidian includes the following steps:
(1) sample treatment:
a. taking silkworm eggs, and adding 10% of KOH by mass percent for treatment to fully soften the egg shells;
b. adding STP lysate, adding glass beads with diameter of 425-;
c, centrifuging at 1000rpm for 2min, and transferring the supernatant into a centrifuge tube;
d. adding protease K with concentration of 20mg/mL and SDS with mass fraction of 10%, and treating at 55 deg.C for 30 min;
e. adding phenol: chloroform: fully and uniformly mixing mixed liquor with the volume ratio of isoamylol of 25:24:1, centrifuging at 12000rpm for 5min, sucking supernatant, placing in a centrifuge tube, and repeating the steps;
f. adding absolute ethyl alcohol into the supernatant, reversing the mixture twice, and centrifuging the mixture at 12000rpm for 5 min;
g. fully pouring out the liquid, pouring the liquid on absorbent paper, standing for 5min, and adding double distilled water to dissolve the DNA to prepare a sample template to be detected;
(2) carrying out PCR amplification on the primer pair for detecting the nosema bombycis, which is processed in the step (1) and is used for obtaining the template to be detected and marked by the nucleic acid molecular marker, wherein the sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.75 and SEQ ID NO.76 or SEQ ID NO.77 and SEQ ID NO. 78;
(3) performing ELISA on the product amplified in the step (2), detecting the OD value at 405nm, detecting the amplified product with the healthy silkworm egg genome as the template as a negative sample, and determining the sample with the OD value 2.1 times larger than the OD value of the negative control as a positive sample; and the sample with the OD value being 2.1 times smaller than that of the negative control is the negative sample.
In the invention, in the step (2), the PCR amplification conditions comprise pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10sec, annealing at 64 ℃ for 15sec, extension at 68 ℃ for 35sec and amplification for 30 cycles; finally, extension was carried out at 68 ℃ for 3 min.
In the invention, in the step (3), the ELISA detection specifically comprises the following steps:
a) adding 1 mu g/mL streptavidin solution into each hole of the ELISA plate, and treating for 3 hours at 4 ℃ overnight or at 18-25 ℃;
b) drying the liquid in the plate, adding PBST into each hole, oscillating for 5min, drying the liquid in the plate, and repeating for four times;
c) diluting the PCR product by 50 times with PBS (phosphate buffer solution) containing 2% fetal calf serum in volume fraction, adding the diluted PCR product into an ELISA plate, repeating the steps for three samples, and incubating for 2 hours at 18-25 ℃;
d) drying the liquid in the plate, adding PBST into each hole, oscillating for 5min, drying the liquid in the plate, and repeating for four times;
e) adding an enzyme-labeled DIG antibody diluent diluted by a PBS solution containing 2% fetal calf serum according to a ratio of 1:1000, and incubating for 45min at 18-25 ℃;
f) drying the liquid in the plate, adding PBST into each hole, oscillating for 5min, drying the liquid in the plate, and repeating for four times;
g) adding 30 percent of H with volume fraction of 1:1000 by volume2O2And PBST solution, and incubating for 15min in dark;
h) a500 mM oxalic acid solution was added for termination, and the OD was measured at 405 nm.
The invention has the beneficial effects that: the invention discloses a kit for detecting silkworm microsporidian by a PCR-ELISA method, which can ensure the specificity of an amplification result by amplifying a proper gene fragment by a marked specific primer pair, thereby ensuring the accuracy of the detection result; performing ELISA on the amplified product, so that a plurality of samples are also detected to realize high flux in the whole detection process on the basis of keeping the objectivity of the detection result; and the screened species-specific primers have important significance for identifying microsporidian infection and the pathogenic epidemiology thereof. Therefore, the invention is an ideal detection means for the nosema bombycis in the eggs of the finished products of the silkworms.
The kit for detecting the nosema bombycis in the silkworm finished eggs provided by the patent further has the following advantages:
high flux: multiple samples can be detected in one experiment, and the ELISA can be operated in running water;
sensitivity: the sensitivity is 10 times higher than that of agarose gel electrophoresis, and silkworm microsporidian which can not be detected in an optical microscope can be detected;
the specificity is high: a large amount of primer screening is carried out to obtain a species-specific primer, and the nosema bombycis can be singly detected; and the specificity of the microsporidian can be distinguished and identified by combining with the genus specificity obtained at the earlier stage.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a diagram showing DNA electrophoresis of a silkworm microsporidian genome amplified with different primers (A: 15 pairs of primers, B: 14 pairs of primers, and C: 15 pairs of primers).
FIG. 2 shows the results of 6 pairs of primer specificity tests (A: OP1-748, J-183, K-245 and M-247; B: U-245 and U-256 pairs of primer specificity tests; N.b represents nosema bombycis; V.n represents nosema nakayae; N.p represents nosema apis; N.a represents nosema bombycis).
FIG. 3 shows the false positive detection of the modified primer.
FIG. 4 shows the sensitivity test (A: primer LUS323 sensitivity; B: M247 sensitivity; P. positive control; N. negative control 10 from left to right6、105、104、103、10210 nosema bombycis, mixed with 50 healthy eggs, respectively).
FIG. 5 shows the detection of silkworm eggs with toxicity (A: LSU 323; B: M247; P. positive control, N. negative control, 1. silkworm eggs with toxicity).
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1 primer screening
Extracting the DNA of the silkworm microsporidian genome by adopting a CTAB method.
Meanwhile, the tussah microsporidian, the bee microsporidian and the Naka amoeba genome are extracted for comparison.
Then according to the comparison of silkworm microsporidian genome with several other microsporidian genomes, screening out the sequence whose similarity is less than 30% to make primer design.
Specific primers are shown in table 1 below.
TABLE 1 primer set for microsporidian detection
Figure BDA0001395634570000041
Figure BDA0001395634570000051
Figure BDA0001395634570000061
The silkworm microsporidian genome DNA is used as a template, the sequence shown in the table 1 is used as a primer for PCR amplification, and the PCR amplification system is shown in the following table 2:
TABLE 2 PCR amplification System
Figure BDA0001395634570000062
Figure BDA0001395634570000071
Amplifying according to the following conditions, and performing pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10sec, annealing at 64 ℃ for 15sec, extension at 68 ℃ for 35sec, 30 cycles, and final extension after 68 ℃ for 3 min. Taking 10 mu L of the amplification product for agarose gel electrophoresis, wherein the conditions of the agarose gel electrophoresis are as follows: 1 XTAE buffer, voltage 180V, electrophoresis time 30min, the results are shown in FIG. 1. The results showed that eight primer pairs, 0P1-748, J183, K245, M247, U245, U256, V220, and V231, were found to be dimer-free and bright in band among the 44 primer pairs designed. Then PCR is carried out by using the nosema bombycis genome diluted by gradient, and the results show that the sensitivity of OP1-748, J183, K245, M247, U245 and U246 is higher, so that six pairs of primers are selected for the next experiment.
Example 2 specificity test
The six pairs of primers selected in example 1 were used to detect the DNA of ① nosema bombycis, ② Naka deformed nosema microsporidian, ③ Antheraea bombycis, ④ Apis nosema DNA, using MightyAmp DNA polymerase Ver.3 from TAKARA, 25. mu.L of the total reaction system, amplified using an Applied Biosystems PCR instrument with the reaction parameters of pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10sec, annealing at 64 ℃ for 15sec, extension at 68 ℃ for 35sec, 30 cycles, extension at 68 ℃ for 3min, 12 ℃ storage, electrophoresis of 10. mu.L of the amplified product in 2% agarose gel electrophoresis, followed by ethidium bromide staining, and ultraviolet lamp observation to determine whether there is a specific band, as shown in FIG. 2, 1-748, K245, M247, U245, U246, five pairs of primers specifically amplifying the microsporidian DNA, and the five pairs of primers are labeled with a DNA marker (DIIN).
Example 3 false Positive detection
In order to detect whether the labeled primer has false positive, the positive uses the DNA of nosema bombycis as a template, and the negative uses ddH2And O is taken as a template, and the amplification is carried out according to the established PCR system. After amplification, 10. mu.L of the mixture was used for agarose gel electrophoresis. The gel electrophoresis conditions were: 1 XTAE buffer solution, voltage 180V, electrophoresis time 30 min. And simultaneously detected by ELISA, and the result is shown in figure 3. The results showed that OP1-748, K245, M247, U246 and LSU323 all gave positive results except the positive result which could not be obtained by U245, and the negative control was close to the blank OD, so that the next experiment could be performed. Wherein LSU323 the concrete sequence is referred to in literature (Niqi, establishment of the test paper strip detection method of bombyx mori corpuscle nucleic acid lateral chromatography [ D)]Chongqing, national focus laboratory of Bombyx mori genome biology, university of southwest, 2016.).
Example 4 sample treatment method
The sample processing method for silkworm microsporidian nucleic acid amplification detection in silkworm finished eggs comprises the following steps:
a. 50 silkworm eggs are taken and put in a 2mL cell freezing tube, and 1mL KOH with the mass fraction of 10 percent is added for treatment for 6min (the treatment time is finely adjusted according to the thickness of the egg shell), so that the egg shell is fully softened.
b. 200 μ L of STP lysate (1 wt% SDS, 1 wt% Triton X-100, 1 wt% Nonidet P40) was added, 3g of 425-600 μm (30-40U.S. sieve) glass beads were added, and the mixture was crushed three times for 5min each in a spore crusher.
c.1000rpm/min for 2min, and the supernatant is transferred to a new 1.5mL centrifuge tube.
d. 20. mu.L of 20mg/mL proteinase K and 30mL of 10% SDS were added and treated at 55 ℃ for 30 min.
e. Add 300. mu.L phenol chloroform (phenol: chloroform: isoamyl alcohol 25:24:1) and mix well, then centrifuge at 12000rpm/min for 5 min. Aspirate the supernatant into a new 1.5mL centrifuge tube and repeat step 5.
f. The supernatant was aspirated and placed in a new 1.5mL centrifuge tube, 300. mu.L of absolute ethanol was added, and after two times of inversion, centrifugation was carried out at 12000rpm for 5 min.
g. The liquid was poured off sufficiently, and the mixture was poured onto absorbent paper, allowed to stand for 5min, and 50. mu.L of double distilled water was added to dissolve the DNA.
Example 5 sensitivity test
M247 and LSU323 were selected among the previously screened primers for further experiments. For detecting the sensitivity of PCR-ELISA, the silkworm microsporidian is diluted to 10 after counting6、105、104、103、102And 10 nosema bombycis, respectively mixing with 50 healthy eggs, treating the sample DNA according to the embodiment 4, and amplifying according to the established PCR system, wherein the positive control takes nosema bombycis DNA as a template, and the negative control takes healthy egg DNA as a template. Amplification ofAfter completion, 10. mu.L of the suspension was subjected to agarose gel electrophoresis. The gel electrophoresis conditions were: 1 XTAE buffer solution, voltage 180V, electrophoresis time 30 min. The results of the simultaneous detection by ELISA are shown in FIG. 4. The results show that the sensitivity of both pairs of primers can reach 103spose/50 silkworm eggs, and the sensitivity of ELISA is increased by 10-100 times compared with the sensitivity of the traditional gel electrophoresis.
Example 6 detection of nosema bombycis
The method for rapidly detecting the nosema bombycis comprises the following steps:
the DNA of the sample to be detected treated in the embodiment 5 is subjected to PCR amplification by using a primer marked by a nucleic acid molecular marker, the DNA of nosema bombycis is used as a template for a positive control, the DNA of healthy silkworm eggs is used as a template for a negative control, and an amplification product is collected; wherein the PCR amplification conditions comprise pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10sec, annealing at 64 ℃ for 15sec, extension at 68 ℃ for 35sec, and amplification for 30 cycles; finally, extension was carried out at 68 ℃ for 3 min.
B. Performing ELISA on the product obtained by amplification in the step A, wherein a sample with OD 2.1 times larger than the OD value of the negative control is a positive sample; the sample with OD 2.1 times smaller than the negative control OD value is the negative sample;
a) adding 100 mu L of streptavidin solution into each hole of the ELISA plate, and standing overnight at 4 ℃ or standing for 3 hours at room temperature;
b) drying the liquid in the plate, adding 200 mu L of PBST into each hole, shaking for 5min, drying the liquid in the plate, and repeating for four times;
c) diluting the PCR product to 50 times with FCS (fetal calf serum)/PBS (2% volume fraction), and adding into enzyme label plate, i.e. adding 98 μ L2% FCS/PBS into 2 μ L PCR product, repeating each sample for three times, and incubating at room temperature for 2 h;
d) drying the liquid in the plate, adding 200 mu L of PBST into each hole, shaking for 5min, drying the liquid in the plate, and repeating for four times;
e) add 100. mu.L enzyme-labeled DIG antibody (1:1000, 2% FCS/PBS), incubate 45min at room temperature;
f) drying the liquid in the plate, adding 200 mu L of PBST into each hole, shaking for 5min, drying the liquid in the plate, and repeating for four times;
g) adding 30% of H2O2And 100. mu.L of ABST solution (1:1000) incubated in the darkCulturing for 15 min;
h) the addition of 50. mu.L oxalic acid solution was stopped, and the absorbance at 405nm was measured, and the results are shown in FIG. 5.
Meanwhile, PCR amplification was performed as a control in the method of example 1, and 10. mu.L of the amplification product was subjected to agarose gel electrophoresis under the conditions: 1 XTAE buffer solution, voltage 180V, electrophoresis time 30 min. Meanwhile, the amplification products were detected by ELISA, and the detection results are shown in FIG. 5.
The result shows that the silkworm eggs and positive plasmids produced by the toxoplasma gondii have positive results, the healthy silkworm eggs have negative results, and the PCR-ELISA sensitivity is higher than that of the PCR method, which indicates that the PCR-ELISA can be used for detecting the nosema bombycis in the silkworm eggs.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
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<213> Artificial sequence (Artificial)
<400>16
gaaccgacgc tttcatagac g 21
<210>17
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>17
tatttagttc ggctttgc 18
<210>19
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>19
acgaggctac ttctgtga 18
<210>19
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>19
attatttagt tcggctttgc 20
<210>20
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>20
acgaggctac ttctgtga 18
<210>21
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>21
ccgctgttat gttggatgt 19
<210>22
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>22
tagccttgag gattgtctt 19
<210>23
<211>22
<212>DNA
<213> Artificial sequence (Artificial)
<400>23
tcaccctgcg ttcaccgctc ta 22
<210>24
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>24
actggttcgc ctcgccttcg 20
<210>25
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>25
tctttggcga tgatgaggtg 20
<210>26
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>26
cggtgaacgc agggtgata 19
<210>27
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>27
tctttggcga tgatgaggtg 20
<210>29
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>29
gcccgcattc ggcgactta 19
<210>29
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>29
ggttcacgct tcgcctatca 20
<210>30
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>30
ttcggtctgg ctgcctttac 20
<210>31
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>31
cggttcacgc ttcgcctat19
<210>32
<211>21
<212>DNA
<213> Artificial sequence (Artificial)
<400>32
gcatccatca cctgccactt a 21
<210>33
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>33
acggttcacg cttcgcctat 20
<210>34
<211>22
<212>DNA
<213> Artificial sequence (Artificial)
<400>34
gcatccatca cctgccactt at 22
<210>35
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>35
gttcacgctt cgcctatcag 20
<210>36
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>36
cacatcggca tccatcacc 19
<210>37
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>37
gcaactacct cgctcgtc 18
<210>38
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>38
gggtttatgt tagcctct 18
<210>39
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>39
tgaagatgcg aatacctc 18
<210>40
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>40
tagccaataa tcgtccag 18
<210>41
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>41
atacagtgcc aggtccata 19
<210>42
<211>22
<212>DNA
<213> Artificial sequence (Artificial)
<400>42
caacataact atggttacta gc 22
<210>43
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>43
atacagtgcc aggtccata 19
<210>44
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>44
tagcgtcatt acccagaac 19
<210>45
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>45
gacagcgacc acagcaaatg 20
<210>46
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>46
taaatgaggc aaaaggcaga 20
<210>47
<211>22
<212>DNA
<213> Artificial sequence (Artificial)
<400>47
tctcaacaat cagaagccat ac 22
<210>48
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>48
ccaccgcata cacgaact 18
<210>49
<211>21
<212>DNA
<213> Artificial sequence (Artificial)
<400>49
ctcaacaatc agaagccata c 21
<210>50
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>50
taccaaccac cgcatacac 19
<210>51
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>51
ccgagtccgc tcagaaag 18
<210>52
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>52
ggcgtggtag aaatgaaaca 20
<210>53
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>53
gccgagtccg ctcagaaa 18
<210>54
<211>21
<212>DNA
<213> Artificial sequence (Artificial)
<400>54
tggcgtggta gaaatgaaac a 21
<210>55
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>55
tccgcttgct aagtttgtg 19
<210>56
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>56
tcaggatagg agccgagtt 19
<210>57
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>57
cgcttgctaa gtttgtgga 19
<210>58
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>58
ctgcgggaaa gattggtgc 19
<210>59
<211>23
<212>DNA
<213> Artificial sequence (Artificial)
<400>59
agtttgtgga gattatggac gag 23
<210>60
<211>17
<212>DNA
<213> Artificial sequence (Artificial)
<400>60
cgctgcggga aagattg 17
<210>61
<211>18
<212>DNA
<213> Artificial sequence (Artificial)
<400>61
atggtttgtc ccgctcct 18
<210>62
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>62
accttaccgc ttggttgatt 20
<210>63
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>63
gcgtcaggtt ggtcaggaag 20
<210>64
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>64
gttgagggtg aggctggaga 20
<210>65
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>65
ggcaaacagt atgctccca 19
<210>66
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>66
aaacgcccgt cagataaaga 20
<210>67
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>67
aaaatgtctg ggaaagttgt 20
<210>68
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>68
tagttctccg ctaaaatgct 20
<210>69
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>69
aaggatggat ttggaggtta 20
<210>70
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>70
ttttgatgcg tattttgagt 20
<210>71
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>71
ggtcagaaag gcggtattg 19
<210>72
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>72
agaacaggaa acagcgtca 19
<210>73
<211>21
<212>DNA
<213> Artificial sequence (Artificial)
<400>73
gataacgact gccagataac c 21
<210>74
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>74
ttccgctaaa tgctgcctc 19
<210>75
<211>22
<212>DNA
<213> Artificial sequence (Artificial)
<400>75
cctcaacaat cagaagccat ac 22
<210>76
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>76
aaccaccgca tacacgaact 20
<210>77
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>77
acaatccacc tcaacaatca 20
<210>78
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>78
ataccaacca ccgcatacac 20
<210>79
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>79
ataatccgtc tcatcaagc 19
<210>80
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>80
agaaacccga catcataac 19
<210>81
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>81
ataatccgtc tcatcaagc 19
<210>82
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>82
aactcaagga aagaaaccc 19
<210>83
<211>20
<212>DNA
<213> Artificial sequence (Artificial)
<400>83
gtcatcaaac accgagtaag 20
<210>84
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>84
agaaagaaac ccgacatca 19
<210>85
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>85
aatcgcagca ttagtggaa 19
<210>86
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>86
aagaaggcgg atgagcaac 19
<210>87
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>87
gaactcaacg gtagacgaa 19
<210>88
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>88
caaatcccga acatccaat 19

Claims (5)

  1. The kit for detecting silkworm microsporidian by the PCR-ELISA method is characterized in that: the kit comprises a primer pair for detecting nosema bombycis marked by a nucleic acid molecular marker, an ELISA reagent for detecting the nucleic acid molecular marker and a sample processing reagent; the sequences of the primer pairs are shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.75 and SEQ ID NO.76 or SEQ ID NO.77 and SEQ ID NO. 78; the ELISA reagent for detecting the nucleic acid molecular marker comprises the following components in concentration: 1 microgram/mL streptavidin containing 2% volume fraction of fetusPBS solution of bovine serum, enzyme-labeled DIG antibody, PBST solution, and H with volume fraction of 30%2O2500mM oxalic acid;
    the sample processing reagent comprises 10% of KOH by mass fraction, STP lysate, glass beads with the diameter of 425 and 600 mu m, 20mg/mL proteinase K, 10% of SDS by mass fraction, phenol: chloroform: mixed solution of isoamylol with volume ratio of 25:24:1 and absolute ethyl alcohol; the concentration of each component of the STP lysate is as follows: 1% mass fraction SDS, 1% mass fraction Triton X-100 and 1% mass fraction Nonidet P40.
  2. 2. The kit for detecting nosema bombycis by PCR-ELISA according to claim 1, wherein: the nucleic acid molecular markers are biotin and digoxin.
  3. 3. The method for detecting nosema bombycis by using the kit according to any one of claims 1 to 2 in non-disease diagnosis, comprising the steps of:
    (1) sample treatment:
    a. taking silkworm eggs, and adding 10% of KOH by mass percent for treatment to fully soften the egg shells;
    b. adding STP lysate, adding glass beads with diameter of 425-; the concentration of each component of the STP lysate is as follows: SDS with the mass fraction of 1%, Triton X-100 with the mass fraction of 1% and Nonidet P40 with the mass fraction of 1%;
    c, centrifuging at 1000rpm for 2min, and transferring the supernatant into a centrifuge tube;
    d. adding protease K with concentration of 20mg/mL and SDS with mass fraction of 10%, and treating at 55 deg.C for 30 min;
    e. adding phenol: chloroform: fully and uniformly mixing mixed liquor with the volume ratio of isoamylol of 25:24:1, centrifuging at 12000rpm for 5min, sucking supernatant, placing in a centrifuge tube, and repeating the steps;
    f. adding absolute ethyl alcohol into the supernatant, reversing the mixture twice, and centrifuging the mixture at 12000rpm for 5 min;
    g. fully pouring out the liquid, pouring the liquid on absorbent paper, standing for 5min, and adding double distilled water to dissolve the DNA to prepare a sample template to be detected;
    (2) carrying out PCR amplification on the primer pair for detecting the nosema bombycis, which is processed in the step (1) and is used for obtaining the template to be detected and marked by the nucleic acid molecular marker, wherein the sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.75 and SEQ ID NO.76 or SEQ ID NO.77 and SEQ ID NO. 78;
    (3) performing ELISA on the product amplified in the step (2), detecting the OD value at 405nm, detecting the amplified product with the healthy silkworm egg genome as the template as a negative sample, and determining the sample with the OD value 2.1 times larger than the OD value of the negative control as a positive sample; and the sample with the OD value being 2.1 times smaller than that of the negative control is the negative sample.
  4. 4. The method of claim 3, wherein: in the step (2), the PCR amplification conditions comprise pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10sec, annealing at 64 ℃ for 15sec, extension at 68 ℃ for 35sec, and amplification for 30 cycles; finally, extension was carried out at 68 ℃ for 3 min.
  5. 5. The method of claim 3, wherein: in the step (3), the ELISA detection specifically comprises the following steps:
    a) adding 1 microgram/mL of streptavidin solution into each hole of the ELISA plate, and treating for 3 hours at 4 ℃ overnight or at 18-25 ℃;
    b) drying the liquid in the plate, adding PBST into each hole, oscillating for 5min, drying the liquid in the plate, and repeating for four times;
    c) diluting the PCR product by 50 times with PBS (phosphate buffer solution) containing 2% fetal calf serum in volume fraction, adding the diluted PCR product into an ELISA plate, repeating the steps for three samples, and incubating for 2 hours at 18-25 ℃;
    d) drying the liquid in the plate, adding PBST into each hole, oscillating for 5min, drying the liquid in the plate, and repeating for four times;
    e) adding an enzyme-labeled DIG antibody diluent diluted by a PBS solution containing 2% fetal calf serum according to a ratio of 1:1000, and incubating for 45min at 18-25 ℃;
    f) drying the liquid in the plate, adding PBST into each hole, oscillating for 5min, drying the liquid in the plate, and repeating for four times;
    g) adding 30 percent of H with volume fraction of 1:1000 by volume2O2And PBST solution, and incubating for 15min in dark;
    h) a500 mM oxalic acid solution was added for termination, and the OD was measured at 405 nm.
CN201710774384.6A 2017-08-31 2017-08-31 Kit for detecting silkworm microsporidian by PCR-ELISA method and detection method thereof Active CN107312869B (en)

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CN101967512A (en) * 2009-07-27 2011-02-09 浙江大学 Fluorescence PCR detection method of pebrine disease and kit thereof
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CN101935707A (en) * 2010-09-03 2011-01-05 浙江省检验检疫科学技术研究院 Nosema bombycis SYBT Green fluorescence quantitative PCR (Polymerase Chain Reaction) detection method as well as kit and specific primer thereof
CN103243170A (en) * 2013-05-24 2013-08-14 江苏省蚕种公司 Detection method of number level of nosema bombycis in silkworm finished-product eggs
CN105154576A (en) * 2015-10-26 2015-12-16 西南大学 Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof

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