CN114277158B - Detection kit for Onchidium chensinensis and application thereof - Google Patents

Abstract

The invention discloses a detection kit and a detection method for rapidly detecting Onchidium chensinensis in environment and fish bodies, and belongs to the technical field of parasite pathogen detection. The invention also discloses a real-time fluorescent quantitative PCR primer for detecting the Oncorhynchus chensinensis, which can quickly detect larvae and imagoes of the Oncorhynchus chensinensis in aquaculture water and fish bodies, greatly improves the detection efficiency, and saves the detection time and the detection cost. The invention has strong specificity, high sensitivity up to 10 copies/mu L, and has no non-specific amplification to the DNA of the culture water environment.

Description

Detection kit for Onchidium chensinensis and application thereof

Technical Field

The invention relates to the field of detection of fish parasite pathogens, relates to a fish pathogen detection kit, and particularly relates to a detection kit for Onchidium chensinensis and application thereof.

Background

The acanthopanax (Acanthosens) is a genus of Echinodermata, Echinodermata and Tetracyclidae, mainly parasitizes fishes, and can bring great economic loss to aquaculture. The spiny beetle can infect various fishes, including eel, pelteobagrus vachelli, south gulf fish, bass, erythroculter, , coilia ectenes, silver carp, bighead carp, crucian carp and the like. At present, most of the spiny worms reported in China are spiny chensinensis (Acanthosentis cheni), in the life history of the spiny chensinensis, arthropods are required to be used as intermediate hosts, and fleas and the like in water environment can be used as the intermediate hosts. Adult fish parasites are mainly parasitized in pyloric caecum of fish, juvenile fish infection can cause large-area death, adult fish infection can cause the change of intestinal microbial structure, the change of specific microbial species composition is caused, the immunity of the fish is reduced, the capability of resisting pathogenic bacteria infection is reduced, and the damage is brought to aquaculture.

At present, the morphology of the Onchidium chensinensis and the morphology of the Onchidium chensinensis is mainly examined by a microscope in China, the methods have the problems of strict requirements on a detection sample (mature males must be obtained and mucus glands of the mature males must be observed), low sensitivity, false positive and the like, and the method for examining the Onchidium chensinensis larvae in a water environment sample by using the microscope is time-consuming, labor-consuming and extremely low in accuracy.

In order to control fish diseases of cultured fishes in time, a technology capable of quickly detecting whether parasites exist in fish bodies or in culture water bodies is urgently needed, so that multiple real-time fluorescent quantitative PCR primers, probes and matched kits for simultaneously detecting three types of spiny worms are provided, and the problems to be solved by technical personnel in the field are urgently needed.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problems of providing a primer combination, a primer pair and a kit for detecting the Oncorhynchus chensinensis, wherein the sensitivity of the primer pair can reach 10 copies/mu L, and the detection kit takes the Oxncorhynchus chensinensis COX2 gene in a sample to be detected as a detection object and has better accuracy and precision.

In order to achieve the purpose, the invention adopts the following technical scheme: a primer combination having:

(I) the upstream primer has the sequence shown in SEQ ID NO:1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9; and

(II) the downstream primer has the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10; or

(III) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I) and/or (II) and has the same or similar functions with the nucleotide sequence shown in (I) and/or (II); or

(IV) and a nucleotide sequence which has at least 80 percent of homology with the nucleotide sequence shown in the (I) and/or (II).

The invention also comprises a primer pair, wherein the primer pair comprises an upstream primer ACCOX2-I-F and a downstream primer ACCOX 2-I-R; the upstream primer ACCOX2-I-F is shown as a sequence SEQ ID NO: 5 is shown in the specification; the downstream primer ACCOX2-I-R has a sequence shown in SEQ ID NO: and 6, respectively.

The invention also comprises the application of the primer combination or the primer pair in the preparation of a reagent or a kit for detecting the thorn fruit worm.

Wherein, the sample detected in the application is fish-like tissue.

The invention also comprises a detection kit of the Oncorhynchus chensinensis, and the detection kit of the Oncorhynchus chensinensis comprises the primer combination or the primer pair.

Preferably, the concentration ratio of the upstream primer to the downstream primer is 3: 4.

Preferably, the detection kit for the Oncorhynchus chensinensis also comprises a positive control and a negative control.

Preferably, the positive control is a recombinant plasmid comprising a nucleic acid sequence as set forth in SEQ ID NO:11, or a fragment thereof.

Preferably, the recombinant plasmid is pBlue-T-COX 2-I.

The kit also comprises a strong positive control with the concentration of 1.0 multiplied by 10⁴Copy/. mu.L of positive plasmid pBlue-T-COX2-I and critical positive control at a concentration of 1.0X10¹Copies/. mu.L of the positive plasmid pBlue-T-COX 2-I.

Preferably, the detection kit further comprises DNA polymerase, DEPC water, real-time fluorescent PCR buffer, dNTP and SYBR green I.

The invention also comprises a detection method based on the detection kit for the thorn worms, and the detection method is a real-time fluorescent quantitative PCR detection method.

Preferably, the reaction system of the real-time fluorescent quantitative PCR detection method adopted by the invention is as follows: 5 pmol/. mu.L of upstream primer ACCOX 2-I-F1.5. mu.L, 5 pmol/. mu.L of downstream primer ACCOX 2-I-R2.0. mu.L, 5. mu.L of sample DNA, 0.6. mu.L of 5U/. mu.L of Taq DNA polymerase, 3. mu.L of DEPC water, and 12.5. mu.L of 2 × inlet real-time fluorescent PCR buffer; the 12.5. mu.L of 2 × import real-time fluorescent PCR buffer was prepared from a mixture of 2.5. mu.L of 10 × buffer from Takara, 2. mu.L of 10mM dNTP, 0.0025. mu.L of 10000 × SYBR green I, and 8. mu.L of DEPC water;

wherein, the PCR reaction conditions are as follows: pollution prevention is firstly carried out at 37 ℃ for 5 min; then pre-denaturation at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40sec for 40 cycles, and fluorescence signal detection was carried out at the end of extension of each cycle.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the invention provides a special primer for fluorescent quantitative PCR detection of the echinacea chensinensis COX2 gene for the first time, so that the fluorescent quantitative PCR detection can be used for detecting the echinacea chensinensis COX2 gene in a sample to be detected.

2. The primer and the kit have extremely high detection sensitivity and specificity, greatly improve the detection efficiency and effectively prevent pollution.

3. Compared with other conventional detection methods of the Oncorhynchus chensinensis, the detection kit and the detection method have the advantages of simple and easy-to-use operation program, can be used for operation programming, and are suitable for large-area popularization and application.

4. The primer pair and the detection kit can evaluate the echinospora chensinensis infection condition of a fish body, and can be used for detecting the echinospora chensinensis in a water body or an environment.

In conclusion, the invention can provide guarantee for rapidly and accurately detecting the Oncorhynchus chensinensis and guarantee for disease prevention, scientific medication and fish health. The special primer and the detection kit can be used for nucleic acid detection of the echinacea chensinensis COX2 gene in fish bodies, water bodies or environments, and have wide application prospects.

Drawings

FIG. 1 is an electrophoretogram of a target gene of Oncorhynchus chensinensis amplified with five pairs of primers, respectively;

FIG. 2 is an electrophoretogram of five pairs of primers amplified target genes of Oncorhynchus chensinensis and six non-target neighbors;

FIG. 3 is a fluorescent amplification curve of 10 Oncorhynchus chensinensis-infected fish and 10 negative samples, note: NC 1-10: negative sample, Pos 1-10: and (4) positive samples.

Detailed Description

The following examples are intended to illustrate the invention in further detail, but are not intended to limit the invention. The experimental methods of the present invention are all referred to the experimental conditions proposed in "molecular biology laboratory Manual of precise edition" (edited by F.M. Osber et al, published by scientific Press 2005).

Example i: primer design and specificity detection for carrying out fluorescence quantitative PCR detection on chondriosome gene of thorn worm

1. Bioinformatics method design primer and primer screening

The mitochondrial whole genome sequence of Onchocera chensinensis (GenBank: KX108947.1) recorded in GenBank is downloaded, and the mitochondrial whole genome sequence of negative control including other Acanthocephala (Acanthocephala) with close classification relation is downloaded, and see Table 1. After comparison is carried out by Clustal X, a proper region is selected to design a Primer, the region is conserved in the Oncorhynchus chensinensis species, but at least 5% -20% of difference exists relative to negative control, and after the region is selected, ABI Primer Express 3.0 real-time fluorescence quantitative PCR Primer design software is adopted to design a synthetic Primer. The same mutation site in the primer allows 2 and less than 2 degenerate bases. Screening the extracted alternative primers according to the following requirements: (1) the length (L) of the primer is between 19 and 28 bp; (2) the Tm value is between 58 and 60 ℃; (3) GC% between 25-75%; (4) polyN is less than or equal to 4 bp; (5) hairpin is less than or equal to 4 bp; (6) coverage > 90%; (7) the length of the amplified product is controlled between 50-250 bp; (8) BLAST screening was performed with specificity scores > Lx0.4. The PCR primer is synthesized by biological engineering (Shanghai) GmbH, the primer requires PAGE purification, the primer is dry powder when the product is delivered, the dry powder is redissolved by sterile water, the content of the dry powder is determined, and the primer is diluted to 100 pmol/mu L of stock solution for later use.

TABLE 1

Negative control species name (Latin name)	GenBank
		Pomphorhynchus rocci	JQ824373.1
Centrorhynchus clitorideus	MT113355.1
		Sphaerirostris picae	MK471355.1
Leptorhynchoides thecatus	AY562383.1
		Brentisentis yangtzensis	MK651258.1
Plagiorhynchus transversus	NC_029767.1
		Centrorhynchus aluconis	KT592357.1
Pallisentis celatus	NC_022921.1

Aiming at the whole genome of the echinacea chensinensis mitochondria, 5 groups of upstream and downstream primers are designed, and the sequences are as follows: ACCOX 1-I:

upstream primer (ACCOX 1-I-F): 5'-GGTGGGTTGGATCTGAGAGA-3'; (100bp)

Downstream primer (ACCOX 1-I-R): 5'-CCAAAAGCCCCCATAAAAAT-3', respectively; ACCOX 1-II:

upstream primer (ACCOX 1-II-F): 5'-TGGCAAGAATTTACGGTTCC-3', respectively; (168bp)

Downstream primer (ACCOX 1-II-R): 5'-CGTAATGGAAATGGGCAACT-3', respectively;

ACCOX2-I：

upstream primer (ACCOX 2-I-F): 5'-GTGGCCAGAAGTGTTGGAGT-3', respectively; (188bp)

Downstream primer (ACCOX 2-I-R): 5'-ATCGCCCAAGAATGAATCAC-3', respectively; ACCOX 3-I:

upstream primer (ACCOX 3-I-F): 5'-GGCATGGTTTCTGAGGGTTA-3', respectively; (114bp)

Downstream primer (ACCOX 3-I-R): 5'-CACTGACCCGTTGGAAGATT-3', respectively; ACCYTB-I:

upstream primer (ACCYTB-I-F): 5'-TATGGTTGGGTGGTTCGTTT-3', respectively; (228bp)

Downstream primer (ACCYTB-I-R): 5'-CCCCACGGTAACACATAACC-3' are provided.

2. Primer specificity detection in molecular biology experiments

2.1 Experimental materials and reagents:

materials: eight echinococcus samples with similar classification statuses are set: the method comprises the steps of setting a negative environment control sample, selecting four breeding ponds which have been subjected to parasite investigation and have negative results, and collecting water samples. The four culture ponds are respectively a grass carp culture pond, a crucian carp culture pond, a yellow catfish culture pond and a coilia ectenes culture pond, the water samples and mud samples of the eight parasites and the four culture ponds are from the provincial water-saving scientific research institute of Hunan province, and the extracted genome is diluted by 10 times in a gradient manner after the genome DNA is extracted for evaluating the specificity and sensitivity of a PCR system.

Reagent: 5U/. mu.L Taq DNA polymerase (Promega, containing 10 × reaction buffer and 25mM Mg^{2 +})、10mM dNTPs(Promega)、DNA marker I(Tiangen)；Millipore H₂Sterilizing under high pressure, packaging, and storing at-20 deg.C.

2.2 primer, Strain testing

The primers designed by the conventional PCR test are used, a PCR reaction system is prepared according to the concentration of each component, and an amplification program is written according to the TM value of the primers and the size of a product. Conventional PCR amplification was performed on a Bio-Rad Mycycler gradient amplification apparatus, and the resulting PCR products were detected by agarose gel electrophoresis in conjunction with a gel imaging system.

2.3 results

2.3.1 specific detection of Oncorhynchus Chensinensis

A. Amplification condition of primer pair positive sample designed by conventional PCR test

The amplification patterns of the five pairs of primers were designed as follows: as can be seen from FIG. 1, for Onchocera chensinensis, the designed five pairs of primers can amplify a target band, which indicates that the self-designed primers and the used PCR amplification system can effectively amplify the target object.

B. Amplification of negative sample by conventional PCR detection system

For the common eight non-target fish parasitic acanthocephalus, the eight water samples and mud samples of the culture pond, the established conventional PCR detection system is negative in detection, and the established conventional PCR detection system is positive in detection, which shows that the established conventional PCR detection system has excellent specificity and can be used for the rapid detection of the acanthocephalus chensinensis (as shown in figure 2).

Example 2: sensitivity detection of fluorescence quantitative primer for chondriosome gene of thorn worm

1. Detection of fluorescent quantitative primer amplification efficiency

1.1 preparation of Positive control and Standard template

The established conventional PCR system is used for amplifying the genome of the thorn worm, and specific target bands corresponding to 5 groups of primers are obtained by 2 percent agarose gel electrophoresis separation. The gel containing the target band was cut with a clean scalpel, and the target DNA was recovered with an agarose gel DNA recovery kit (Tiangen Biochemical technology (Beijing) Ltd.). The recovered partial DNA was added to a ligation system (10. mu.L, containing the linear cloning vector pBlue-T (2984bp), ligase and ligase buffer) and ligated overnight at 16 ℃. The next day, all the ligations were transformed into prepared E.coli competent cells, spread on LB plates containing ampicillin, and cultured at 37 ℃ until colonies grew. Colonies were picked and prepared as a suspension, verified by PCR using the vector primers and colonies and positive clones were selected. Bacteria corresponding to the positive clones are cultured in an LB liquid culture medium overnight, and 1mL of extracted plasmid DNA (common plasmid miniextraction kit, Tiangen Biochemical technology (Beijing) Co., Ltd.) is taken, so that positive plasmids are obtained, wherein the positive plasmids corresponding to different primers are different. The concentration of the extracted positive plasmid was measured and diluted to 100 ng/. mu.L in bulk to serve as a positive control for PCR detection.

1.2 primer PCR amplification efficiency detection

1.2.1 gradient template preparation

The diluted positive control plasmid of 1.1 is diluted by sterile water in a gradient of 10 times and used as a template for optimizing a fluorescent quantitative PCR reaction system. Get 10^-4、10^-5、10^-6、10^-7、10^-8、10^-9The dilution numbers correspond to L1, L2, L3, L4, L5 and L6 in sequence. Subpackaging and storing at-70 deg.C for use.

1.2.2 fluorescent quantitative PCR buffer and PCR program

The PCR reaction system is as follows: 1.5 μ L (5 pmol/. mu.L) of the upstream primer, 1.5 μ L (5 pmol/. mu.L) of the downstream primer, 5 μ L of sample DNA, 0.6 μ L of Taq DNA polymerase (5U/. mu.L), 4 μ L of DEPC water, 12.5 μ L of 2 × 12.5 μ L of inlet real-time fluorescent PCR buffer; 12.5 u L2 x import real-time fluorescence PCR buffer solution Takara 10 x buffer, 2 u L10 mM dNTP, 0.0025 u L10000 x SYBR green I and 8 u LDEPC water mixture preparation; the PCR reaction conditions are as follows: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturation at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40 cycles, and fluorescence signal detection was carried out at the end of extension in each cycle, and the results are shown in Table 2.

1.2.3 results

TABLE 2

Primer and method for producing the same	10-4	10-5	10-6	10-7	10-8	Efficiency of amplification
							ACCOX1-I	16.4	18.2	22.1	25.2	27.3	122.5％
ACCOX1-II	14.5	18.7	21.6	25.2	29.1	90.6％
							ACCOX2-I	17.0	19.9	23.6	26.8	30.2	99.7％
ACCOX3-I	16.4	19.6	23.4	26.3	30.5	93.4％
							ACCYTB-I	17.2	20.1	24.2	27.5	31.6	88.9％

Note: the values are indicated in the table as Ct values.

2. Optimization of fluorescent quantitative PCR primer dosage

With diluted L2 (10)^-5) The positive control DNA is used as a template for optimizing the amount of the primer, and the amounts of the upstream and downstream of the primer are respectively optimized by gradient dilution within the range of 5.0-12.5pmol, wherein the primer combination is a combination A of 1.0 mu L multiplied by 5 pmol/mu L and 2.0 mu L multiplied by 5 pmol/mu L; combination B1.0. mu.L.times.5 pmol/. mu.L and 2.5. mu.L.times.5 pmol/. mu.L; combination C1.5. mu.L.times.5 pmol/. mu.L and 2.0. mu.L.times.5 pmol/. mu.L; combination D1.5. mu.L.times.5 pmol/. mu.L and 1.5. mu.L.times.5 pmol/. mu.L; combination E2.0. mu.L.times.5 pmol/. mu.L and 2.5. mu.L.times.5 pmol/. mu.L; combination F2.5 muL.times.5 pmol/. mu.L and 1.5. mu.L.times.5 pmol/. mu.L. The PCR reaction system is as follows: sample DNA 5. mu.L, Taq DNA polymerase (5U/. mu.L) 0.6. mu.L, real-time fluorescent PCR buffer (2X) 12.5. mu.L (2.5. mu.L of 10 XBuffer from Takara, 2. mu.L of 10mM dNTP and 0.0025. mu.L of a mixture of 10000 XSSYBR green I and 8. mu.L DEPC water) and sterile water to 25. mu.L. The PCR reaction conditions are as follows: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturation at 95 ℃ for 3 min; finally, 95 ℃ lOsec and 60 ℃ 40sec were amplified for 40 cycles, and fluorescence signal detection was performed at the end of extension of each cycle. As shown in Table 3, it was confirmed that the primers worked well in the reaction of 5.0 to 12.5pmol, and that ACCOX2-I primer showed the highest amplification efficiency (the lowest average Ct value) at the upstream primer amount of 1.5. mu.L.times.5 pmol/. mu.L and the downstream primer amount of 2.0. mu.L.times.5 pmol/. mu.L (combination C). The optimal primer combination of ACCOX1-I is combination C, the optimal primer combination of ACCOX1-II is combination A, the optimal primer combination of ACCOX2-I is combination C, the optimal primer combination of ACCOX3-I is combination D, and the optimal primer combination of ACCYTB-I is combination A.

TABLE 3

Primer and method for producing the same	ACCOX1-I	ACCOX1-II	ACCOX2-I	ACCOX3-I	ACCYTB-I
						Combination A	20.2	21.3	19.3	19.8	20.3
Combination B	19.5	21.6	19.0	19.7	21.1
						Combination C	19.3	21.5	18.5	19.8	20.8
Combination D	19.6	22.0	19.2	19.6	21.6
						Combination E	19.4	22.1	18.9	20.1	21.5
Combination F	19.6	22.6	19.3	20.3	21.2

Note: the average Ct values are noted in the tables.

3. Fluorescent quantitative primer sensitivity detection

And (3) carrying out fluorescent quantitative PCR detection by taking the positive control plasmid DNA as a template to establish a standard curve. The specific operation is as follows: plasmid concentration (in ng/. mu.L) was first accurately measured using a qubit4.0 fluorescence quantifier using the formula: copies/. mu.L ═ 6.02X 10²³) X (concentration ng/. mu.L.times.10)^-9) /(DNA length. times.660) the copy number concentration of the original plasmid (in copies/. mu.L or copies/. mu.L) was calculated, and then all positive plasmids were uniformly diluted to 1.0X10¹¹Copy/. mu.L, after which the plasmid DNA was serially diluted 10-fold to I: 1.0X10¹⁰Copy/. mu.L; II: 1.0X10⁹Copy/. mu.L; III: 1.0X10⁸Copy/. mu.L; IV: 1.0X10⁷Copy/. mu.L; v: 1.0X10⁶Copy/. mu.L; VI: 1.0x10⁵Copy/. mu.L; VII: 1.0X10⁴Copy/. mu.L; VIII: 1.0X10³Copy/. mu.L; IX: 1.0X10²Copy/. mu.L; x: 1.0X10¹Copy/. mu.L; XI: 1.0X10⁰Copies/. mu.L. Parallel experiments were repeated 3 times for each dilution. The PCR reaction system for detecting the standard substance is as follows: the primer concentrations were selected from the optimal combination concentration of each primer in the previous experiment, 5. mu.L of plasmid DNA, 0.6. mu.L of Taq DNA polymerase (5U/. mu.L), 12.5. mu.L of real-time fluorescent PCR buffer (2X) (prepared from 10 XBuffer 2.5. mu.L from Takara, 2. mu.L of 10mM dNTP and 0.0025. mu.L of a mixture of 10000 XSSYBR green I and 8. mu.L of DEPC water), and supplemented with sterile water to 25. mu.L. The PCR reaction conditions are as follows: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturation at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40sec for 40 cycles, and fluorescence signal detection was carried out at the end of extension of each cycle.

The lowest plasmid concentration with positive result (Ct <40) is taken as the detection sensitivity, and 0.03 normalized fluorescence unit (norm. Fluoro) is uniformly taken as a critical value when a fluorescence curve is analyzed to calculate the Ct value in order to ensure that the result has comparability. For data analysis, the plasmid concentration was taken as log10, and the log10 copies/. mu.L value of each primer was calculated, and the detection results of each primer are shown in Table 4 below, wherein the higher the value, the higher the lowest concentration that can be detected, and the lower the value, the lower the lowest concentration that can be detected, i.e., the higher the sensitivity.

TABLE 4

The results in Table 4 show that the primer pair ACCOX2-I for the Onychia chensinensis has 8 magnitude linear detection ranges under the condition of specific primer combination, the sensitivity is extremely high, and the minimum detection concentration can reach 10 copies/mul.

According to the results of sensitivity and amplification efficiency, the third primer pair ACCOX2-I-F/ACCOX2-I-R was selected to continue the subsequent experiments. The PCR fragment obtained by amplifying the third primer pair ACCOX2-I-F/ACCOX2-I-R is named COX2-I, the sequence is shown as SEQ ID NO:11, the positive control plasmid obtained by constructing according to 1.1 of the example 2 is pBlue-T-COX2-I, and the basic composition and the content of each component of the real-time fluorescent quantitative PCR detection reagent are shown in the following table 5.

TABLE 5

Example 3: detection of actual sample by established fluorescent quantitative system

1. Real-time fluorescent quantitative PCR method for detecting 20 parts of fish samples

1.1 extraction of DNA from Fish-like tissue

10 samples of diseased and suspected coilia ectenes and 10 samples of healthy coilia ectenes were collected and DNA was extracted rapidly by the chelex-100(Bio-Rad) boiling method. The method comprises the following steps: a small piece of the cecal tissue was taken and 200. mu.L of Millipore H was added₂O, mashing, removing the unsteamed tissue mass, centrifuging the remaining turbid solution at 12,000 rpm for 1min, discarding the supernatant, and adding 200. mu.L of Millipore H₂And (4) resuspending the solution. Mixing well, adding 50 μ L into 200 μ LAnd (2) in 6% of chelex-100, uniformly mixing, preserving heat for 20min at 56 ℃, shaking vigorously, uniformly mixing, preserving heat for 8min at 100 ℃, shaking violently, centrifuging for 3min at 12,000, taking the supernatant as a PCR template, and detecting by using an established conventional PCR detection system. The remaining template was stored at-20 ℃ for review.

1.2 detection of DNA samples from diseased Fish by the methods established in examples 1, 2 and 3

The PCR reaction system for detecting diseased fish is as follows: 1.5 μ L (5 pmol/. mu.L) of the forward primer, 2.0 μ L (5 pmol/. mu.L) of the reverse primer, 5 μ L of plasmid DNA (pBlue-T-COX2-I), 0.6 μ L of 5U/. mu.L Taq DNA polymerase, 3 μ L DEPC water, 12.5 μ L of 2 × 12.5 μ L of real-time fluorescent PCR buffer: 12.5 μ L of 2 × import real-time fluorescent PCR buffer was prepared from a mixture of 2.5 μ L of 10 × buffer from Takara, 2 μ L of 10mM dNTP, 0.0025 μ L of 10000 × SYBR green I, and 8 μ L of DEPC water. The PCR reaction conditions were: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturing at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40sec for 40 cycles, and fluorescence signal detection was carried out at the end of extension of each cycle. The results show that: when the DNA of 10 confirmed samples of the fish infected with Onychira chensinensis is used as a template, a target fluorescence amplification curve appears, and when the DNA of the rest 10 samples is used as a template, no amplification curve appears (see FIG. 3).

Example 4: stability detection of fluorescence quantitative PCR detection kit for echinococcus chensinensis COX2 gene

The fluorescence quantitative PCR detection kit for the echinacea chensinensis COX2 gene comprises 1 ml/tube of echinacea chensinensis reaction liquid and 30 mu L/tube of Taq polymerase (5U/mu L, 0.6 mu L of each reaction) which are independently packaged, and the two reagent tubes are jointly packaged in an outer packaging box. Wherein the reaction solution contains dNTPs and MgC1₂SYBR green I and primer mixture prepared from 12.5. mu.L of 2 XBuffer (prepared from 10 XBuffer 2.5. mu.L, 2. mu.L of 10mM dNTP and 0.0025. mu.L of a mixture of 10000 XSSYBR green I and 8. mu.L DEPC water available from Takara), 1.5. mu.L of upstream primer and 2.0. mu.L of downstream primer in the primer combination C determined in example 2, multiplying each component by a coefficient (such as 10000, according to the production), mixing, and packaging, wherein each 1mL is convenient for detection, and the kit can also compriseComprising a strong positive control (10) separately packaged^-5Dilution of positive control plasmid (pBlue-T-COX2-I) L2, Critical positive control (10)^-8The diluted positive control plasmid (pBlue-T-COX2-I) L5) and DNA extracting solution (formula: chelex-100) and a negative control (sterile water), and are assembled in an external packaging box together with the thorn worm reaction liquid and a Taq polymerase reagent tube. And (3) PCR reaction system: 1.5. mu.L of the upstream primer (5 pmol/. mu.L), 2.0. mu.L of the downstream primer (5 pmol/. mu.L), 5. mu.L of the sample DNA, 0.6. mu.L of Taq DNA polymerase (5U/. mu.L), 12.5. mu.L of real-time fluorescent PCR buffer (2X) (prepared with 10 XBuffer 2.5. mu.L, 2. mu.L of 10mM dNTP from Takara, and 0.0025. mu.L of a mixture of 10000 XSSYBR Green I and 8. mu.L of DEPC water), and supplemented with sterile water to 25. mu.L. The PCR reaction conditions are as follows: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturation at 95 ℃ for 3 min; finally, 95 ℃ lOsec and 60 ℃ 40sec were amplified for 40 cycles, and fluorescence signal detection was performed at the end of extension of each cycle. The results show (Table 6, the results are stable after 5 times of repeated determination, and the positive control determined as the kit of the invention is pBlue-T-COX 2-I.

TABLE 6 test results of Positive controls

Repeat (R) to	1	2	3	4	5
						Ct value	22.4	22.9	22.1	23.0	22.5

Example 5: performance evaluation of fluorescent quantitative PCR detection kit for echinococcus chensinensis COX2 gene

In order to evaluate the performance of the kit, small samples of products are prepared for many times according to the optimized process of the products, and clinical tests are carried out on the sensitivity, specificity, accuracy and stability of the kit so as to examine the performance of the products.

1. Linear range and sensitivity test for kit detection

The positive plasmid pBlue-T-COX2-I (original concentration is 1.0X 10) of the COX2 gene of the thorn worm⁹Copies/. mu.L) were serially diluted 10-fold to 10 with a DEPC aqueous dilution^-9Namely, sensitivity control (take L1 (10)^-4Dilution), L2 (10)^-5Dilution), L3 (10)^-6Dilution), L4 (10)^-7Dilution), L5 (10)^-8Dilution), L6 (10)^-9Dilution) as a quality control for evaluating the sensitivity of the kit, numbers L1-L6. Subpackaging and storing at-70 deg.C for use. ) The kit of the invention is used for detection. And (3) PCR reaction system: 1.5. mu.L (5 pmol/. mu.L) of the upstream primer, 2.0. mu.L (5 pmol/. mu.L) of the downstream primer, 5. mu.L of sample DNA, 0.6. mu.L of Taq DNA polymerase (5U/. mu.L), 12.5. mu.L of real-time fluorescent PCR buffer (2X) (prepared with 10 XBuffer 2.5. mu.L from Takara, 2. mu.L of 10mM dNTP and 0.0025. mu.L of a mixture of 10000 XSSYBR green I and 8. mu.L of LDEPC water), and 25. mu.L of sterile water was supplemented. The PCR reaction conditions are as follows: pollution prevention is firstly carried out at 37 ℃ for 5 min; then pre-denaturation at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40sec for 40 cycles, and detection of a fluorescent signal was carried out at the end of extension of each cycle. The results (Table 7) show that 10^-9Dilution (L6) sample could not be detected, at 10^-8Since the dilution (L5) of the kit is usually detectable, the kit of the present invention contains 10 or less of the above-mentioned compounds^-8Dilution (concentration 1.0X 10)¹Copy/mu L) of the echinacea chensinensis COX2 gene, has higher sensitivity. Sensitivity and linear analysis are carried out on small samples of products in different batches, and the result shows that the primers can be stably measured out of 10^-8Plasmid samples at dilution, for 10^-9Dilution of the sample, the kit of the present invention cannot be detected. Therefore, it is 10^-8Dilution (concentration 1.0X 10)¹Copy/. mu.L) was the lowest detected value.

TABLE 7 test results for the linear range of the kit of the invention

Dilution gradient	10-4	10-5	10-6	10-7	10-8	10-9
							Ct value	19.4	22.5	26.3	29.4	32.5	No Ct

2. Specificity analysis of kit detection

And detecting 8 main fish echinococcus by adopting small samples of products in different batches. And (3) PCR reaction system: 1.5 μ L (5 pmol/. mu.L) of the forward primer, 2.0 μ L (5 pmol/. mu.L) of the reverse primer, 5 μ L of sample DNA, 0.6 μ L of 5U/. mu.LTaq DNA polymerase, 3 μ L of DEPC water, 12.5 μ L of 2 × inlet real-time fluorescent PCR buffer; 2 × import real-time fluorescent PCR buffer was prepared from 2.5 μ L of Takara 10 × buffer, 2 μ L of a mixture of 10mM dNTP, 10000 × SYBR green I0.0025 μ L, and 8 μ L of DEPC water. The PCR reaction conditions were: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturation at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40sec for 40 cycles, and fluorescence signal detection was carried out at the end of extension of each cycle. The result (Table 8) shows that no Ct value is detected in N1-NIO, which proves that the kit of the invention has good specificity.

TABLE 8 test results of the specificity of the kit of the present invention

Sample (I)

N1

N2

N3

N4

N5

N6

N7

N8

Ct value

No Ct

No Ct

No Ct

No Ct

No Ct

No Ct

No Ct

No Ct

3. Precision of kit batch detection

The use of a precision quality control (concentration of plasmid pBlue-T-COX 2-I1.0X 10)⁹Copy/. mu.L dilution to 1.0X10^-5Copy/. mu.L as a precise quality control substance) for quality control of the DNA detection kit for Onchocera chensinensis. And (3) respectively carrying out 10 times of repeated detection on the reaction system, wherein the PCR reaction system comprises: 1.5. mu.L of the upstream primer (5 pmol/. mu.L), 2.0. mu.L of the downstream primer (5 pmol/. mu.L), 5. mu.L of the sample DNA, 0.6. mu.L of Taq DNA polymerase (5U/. mu.L), 12.5. mu.L of real-time fluorescent PCR buffer (2X) (prepared with 10 XBuffer 2.5. mu.L, 2. mu.L of 10mM dNTP from Takara, and a mixture of 10000 × SYBR Green I0.0025. mu.L and 8. mu.L of DEPC water), and supplemented with sterile water to 25. mu.L. The PCR reaction conditions are as follows: pollution prevention is firstly carried out for 5min at 37 ℃; then pre-denaturation at 95 ℃ for 3 min; finally, amplification was carried out at 95 ℃ for 10sec and 60 ℃ for 40sec for 40 cycles, and fluorescence signal detection was carried out at the end of extension of each cycle. CV% of Ct values of 10 precision quality control products<10 percent, the kit of the invention is proved to have good precision.

4. Determination of the accuracy of the kit

The accuracy of the kit of the invention is confirmed by a sequencing method. Sequencing the amplified product, wherein the sequence is completely consistent with an expected result, and the detection result of the kit is proved to be accurate.

5. Stability assay for kits

(1) Stability of the kit

The stability of the product depends on the stability of the individual components. The real-time fluorescence quantitative PCR reaction solution, Taq DNA polymerase and positive reference substance are stored at-20 ℃ in a laboratory, and are taken out and stored in a refrigerator at 4 ℃ all the time, and the performance of the PCR reaction solution, the Taq DNA polymerase and the positive reference substance is not reduced after the PCR reaction solution, the Taq DNA polymerase and the positive reference substance are stored for one week.

In the product transportation prepared for clinical tests, a whole set of products (comprising real-time fluorescence quantitative PCR reaction solution, Taq DNA polymerase and kit reference products) are subjected to a series of back and forth processes of freezing at-20 ℃, long-distance transportation at 4 ℃, freezing at-20 ℃, re-fusion and the like for 3 days in advance, and quality control products are used for detection, so that the detection results have no significant difference. The components of the kit of the invention are shown to be quite stable.

(2) Stability of control

The stability of the reference substance has great influence on the analysis and judgment of the test result, and the reference substance of the kit is mainly used for controlling the quality of the reaction system. A strong positive control (1.0X 10 concentration) was used in the kit⁴Copy/. mu.L of positive plasmid pBlue-T-COX2-I), one-part critical positive (concentration 1.0X 10)¹Copies/. mu.L of the positive plasmid pBlue-T-COX2-I) and a negative control (sterile water) were subjected to a freeze-thaw test. The experimental results are shown in table 9, and the results show that the reference substance in the kit of the present invention also has good stability.

TABLE 9 Freeze-thaw test results for positive controls

Number of freeze thawing	CT value of positive control	CT value of Critical Positive control	CT value of negative control
				1	22.3	32.3	No Ct
2	22.4	32.7	No Ct
				3	21.8	32.8	No Ct
4	22.3	32.6	No Ct
				5	22.2	32.8	No Ct
6	21.8	32.4	No Ct
				7	22.1	32.5	No Ct
8	22.3	32.3	No Ct

Sequence listing

<110> research institute of Water-saving and Productivity science in Hunan province

<120> detection kit for Onchidium chensinensis and application thereof

<141> 2021-12-22

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> ACCOX1-I-F(Artificial Sequence)

<400> 1

ggtgggttgg atctgagaga 20

<210> 2

<211> 20

<212> DNA

<213> ACCOX1-I-R(Artificial Sequence)

<400> 2

ccaaaagccc ccataaaaat 20

<210> 3

<211> 20

<212> DNA

<213> ACCOX1-II-F(Artificial Sequence)

<400> 3

tggcaagaat ttacggttcc 20

<210> 4

<211> 20

<212> DNA

<213> ACCOX1-II-R(Artificial Sequence)

<400> 4

cgtaatggaa atgggcaact 20

<210> 5

<211> 20

<212> DNA

<213> ACCOX2-I-F(Artificial Sequence)

<400> 5

gtggccagaa gtgttggagt 20

<210> 6

<211> 20

<212> DNA

<213> ACCOX2-I-R(Artificial Sequence)

<400> 6

atcgcccaag aatgaatcac 20

<210> 7

<211> 20

<212> DNA

<213> ACCOX3-I-F(Artificial Sequence)

<400> 7

ggcatggttt ctgagggtta 20

<210> 8

<211> 20

<212> DNA

<213> ACCOX3-I-R(Artificial Sequence)

<400> 8

cactgacccg ttggaagatt 20

<210> 9

<211> 20

<212> DNA

<213> ACCYTB-I-F(Artificial Sequence)

<400> 9

tatggttggg tggttcgttt 20

<210> 10

<211> 20

<212> DNA

<213> ACCYTB-I-R(Artificial Sequence)

<400> 10

ccccacggta acacataacc 20

<210> 11

<211> 188

<212> DNA

<213> COX2-I(Artificial Sequence)

<400> 11

gtggccagaa gtgttggagt agttgggtgt cagtggtatt ggttgtatac tatgggttct 60

atagttgtta gatcttaccc agaaggcggg tttcgactgt tagatgtgga ttgtcgttta 120

gttatcaatt ctggtttgtg gtataaaatt tatattactt caaatgacgt gattcattct 180

tgggcgat 188

Claims (8)

1. The application of a primer pair in preparing a kit for detecting the thorn fruit worm, wherein the primer pair comprises an upstream primer ACCOX2-I-F and a downstream primer ACCOX 2-I-R; the sequence of the upstream primer ACCOX2-I-F is shown as SEQ ID NO: 5 is shown in the specification; the sequence of the downstream primer ACCOX2-I-R is shown as SEQ ID NO: and 6.

2. The use of claim 1, wherein the sample to be tested in said use is fish-like tissue.

3. The detection kit for the thorn fruit worm is characterized by comprising a primer pair, wherein the primer pair is an upstream primer ACCOX2-I-F and a downstream primer ACCOX 2-I-R; the sequence of the upstream primer ACCOX2-I-F is shown as SEQ ID NO: 5 is shown in the specification; the sequence of the downstream primer ACCOX2-I-R is shown as SEQ ID NO: and 6.

4. The detection kit for Onchidium chensinensis according to claim 3, wherein the concentration ratio of the upstream primer to the downstream primer in the primer pair is 3: 4.

5. The detection kit for Onchidium chensinensis according to claim 3, wherein: the detection kit for the Oncorhynchus chensinensis also comprises a positive control and a negative control.

6. The detection kit for Onchidium chensinensis according to claim 3, wherein: the positive control is a recombinant plasmid comprising the nucleic acid sequence set forth in SEQ ID NO:11, or a fragment of DNA as set forth in fig. 11.

7. The detection kit for Onchidium chensinensis according to claim 3, wherein: the detection kit also comprises DNA polymerase, DEPC water, real-time fluorescent PCR buffer solution, dNTP and SYBR green I.

8. The detection kit for Onchidium chensinensis according to claim 3, wherein: the detection kit further comprises a strong positive control: the concentration is 1.0 × 10⁴Copies/. mu.L of the positive plasmid pBlue-T-COX2-I, and Critical positive controls: the concentration is 1.0 × 10¹Copies/μ L of the positive plasmid pBlue-T-COX 2-I; the positive plasmid pBlue-T-COX2-I comprises the nucleotide sequence shown in SEQ ID NO:11, or a fragment of DNA as set forth in fig. 11.

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