CN116219045A - Eriocheir sinensis helicobacter MIRA and MIRA-LFD detection method and application thereof - Google Patents
Eriocheir sinensis helicobacter MIRA and MIRA-LFD detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a detection method of Eriocheir sinensis helicobacter sp MIRA and MIRA-LFD and application thereof, wherein the MIRA technology has lower requirements on samples and laboratory environment, can amplify a small amount of nucleic acid molecules to detectable level, and combines with a lateral flow chromatography test strip (LFD) to form a MIRA-LFD rapid diagnosis method; meanwhile, the methods of the Eriocheir sinensis helicobacter MIRA and MIRA-LFD have high specificity, and the sensitivity performance is stronger than that of PCR; meanwhile, the detection methods of the MIRA and the MIRA-LFD of the Eriocheir sinensis are rapid, sensitive, high in specificity, free of special instruments and the like, are suitable for rapid detection of the Eriocheir sinensis and can be suitable for clinical sample detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection method of Eriocheir sinensis helicobacter MIRA and MIRA-LFD and application thereof.
Background
Eriocheir sinensis (commonly called river crab) plays an important role in the fresh water aquaculture in China, and has high economic value and nutritional value. Along with the rapid and large-scale development of Eriocheir sinensis cultivation, the environmental condition of the water body is rapidly deteriorated, diseases frequently occur and are more serious, wherein the serious diseases are river crab tremble diseases, and huge losses are caused to the Eriocheir sinensis cultivation industry. In recent years, the pathogenesis of the trembling disease of river crabs has been unknown, some are considered to be viruses, some are considered to play an important role in bacteria and living environment, and other people propose that pathogens are rickettsia-like microorganisms, until Wang Wen and the like in 2003, the pathogens are separated from the ill eriocheir sinensis for the first time, and the pathogens are named as eriocheir sinensis spiroplasma by research in aspects of system taxonomy, serology and the likeSpiroplasma eriocheiris, Se）。
At present, common detection methods for detecting the helicobacter eri of Eriocheir sinensis comprise a western blot method, a common PCR method, an RT-PCR method and an LAMP method, but the methods have the defects of easy pollution, complicated operation, moderate sensitivity, non-specific amplification, long time consumption, special instrument and equipment requirement and the like. Therefore, a detection method of the Eriocheir sinensis helicobacter sp, which is simple to operate and high in specificity, is needed.
Disclosure of Invention
In order to solve the problems, the invention discloses a detection method of Eriocheir sinensis mycoplasma MIRA and MIRA-LFD and application thereof, wherein the MIRA technology has lower requirements on samples and laboratory environment, can amplify a small amount of nucleic acid molecules to detectable level, and combines with a lateral flow chromatography test strip (LFD) to form a MIRA-LFD rapid diagnosis method; meanwhile, the methods of the Eriocheir sinensis helicobacter MIRA and MIRA-LFD have high specificity, and the sensitivity performance is stronger than that of PCR; meanwhile, the detection methods of the MIRA and the MIRA-LFD of the Eriocheir sinensis are rapid, sensitive, high in specificity, free of special instruments and the like, are suitable for rapid detection of the Eriocheir sinensis and can be suitable for clinical sample detection.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a detection method of Eriocheir sinensis helicobacter MIRA and MIRA-LFD comprises the following steps:
s1, synthesizing and screening a primer and a probe;
s2, establishing a MIRA reaction system;
s3, establishing a MIRA-LFD reaction system;
s4, specific detection.
As an improvement of the present invention, the specific operation steps of the step S1 are as follows:
(1) Designing a primer: using 30-35bp primer without secondary structure, amplifying fragment size 150-300bp, designing 4 pairs of primers by Olige6 software;
(2) Primary screening: designing a sequence with the length of 46-52nt and complementary to a target fragment in the middle of an upstream primer and a downstream primer, modifying a FAM at the 5 end, marking a dSpacer (tetrahydrofuran, THF) at the middle position of the 5 'end and the 3' 'end, marking a modification group (C3 spacer) at the 3' end as a recognition site of nfo, and designing 2 pairs of probe primers by Olige6 software;
(3) Final screening: and finally screening the two groups of primers to obtain 1 group of 292F/446R primers with the fragment size of about 186bp and the expected size being consistent for detection by a follow-up MIRA method, and designing a group of probe primers for detection by the follow-up MIRA-LFD method by using the fragment.
As an improvement of the present invention, the specific operation steps of the step S2 are as follows:
(1) 25. Mu.L of MIRA reaction system was established, 29.4. Mu.L of a dissolution Buffer (A Buffer) was added to the lyophilized mixed enzyme, and the mixture was divided into 2 portions;
(2) 1. Mu.L of Eriocheir sinensis helicobacter DNA, 6.05. Mu.L of sterile water, 1. Mu.L of 10. Mu. Mol/L forward primer, 1. Mu.L of 10. Mu. Mol/L reverse primer and 1.25. Mu.L of magnesium acetate (B Buffer) are added to each portion;
(3) Mixing the mixture uniformly, vibrating for 1s, centrifuging for 1s, repeating for two times, and immediately placing the mixture into a water bath kettle for reaction to obtain a MIRA product;
(4) The MIRA product was purified by phenol: chloroform: isopropanol=25: 24:1 followed by analysis by 2% agarose gel electrophoresis.
As an improvement of the present invention, the specific operation steps of the step S3 are as follows:
(1) Establishing a 25 mu L MIRA-LFD reaction system, adding 29.4 mu L dissolving Buffer (A Buffer) into the freeze-dried mixed enzyme, and dividing the mixture into 2 parts;
(2) 1. Mu.L of Eriocheir sinensis helicobacter DNA, 6.85. Mu.L of sterile water, 0.5. Mu.L of forward primer of 1. Mu.mol/L, 0.5. Mu.L of reverse primer of 1. Mu.mol/L, and 1.25. Mu.L of magnesium acetate (B Buffer) are added to each portion;
(3) Mixing the mixture uniformly, vibrating for 1s, centrifuging for 1s, repeating for two times, and immediately placing the mixture into a water bath kettle for reaction to obtain a MIRA-LFD product;
(4) 10. Mu.L of the reaction product was placed in a 20. Mu.L PCR cuvette, 95. Mu.L of sterile, sterile water was added thereto, mixed well, and a lateral chromatographic strip was inserted, allowed to stand for 5min, and the result was observed.
As an improvement of the present invention, the specific operation steps of the step S4 are as follows: the screened optimal primer and certain reaction conditions are used, and DNA of bacillus subtilis, bacillus thuringiensis, staphylococcus aureus, escherichia coli, aeromonas hydrophila, salmonella and eriocheir sinensis spiroplasma are used as templates, and a blank control is set at the same time to perform MIRA and MIRA-LFD detection.
As an improvement of the present invention, the reaction conditions are specifically: the reaction temperature of MIRA and MIRA-LFD is 37 ℃ and the reaction time is 10min.
The invention also provides application of the detection method in the Eriocheir sinensis helicobacter.
The invention also provides application of the detection method in clinical sample detection.
The beneficial effects of the invention are as follows:
the invention establishes a MIRA and MIRA-LFD detection method, does not need special instruments, has the advantages of rapidness, sensitivity, strong specificity and the like, can be used for detecting whether the eriocheir sinensis carries pathogenic spiroplasma, timely takes measures, avoids pathogen diffusion, reduces the loss caused by the cultivation of the eriocheir sinensis, and promotes the healthy development of the eriocheir sinensis cultivation industry.
Drawings
FIG. 1 is a primer screen of the invention, wherein: "-" indicates a negative control using sterile water as a template, "+" indicates using Eriocheir sinensis helicobacter as a template, and positive control is a kit with the template and the primer.
FIG. 2 is an optimization of the MIRA reaction temperature and time of the present invention, wherein: a is reaction temperature optimization, and b is reaction time optimization.
FIG. 3 is an optimization of the reaction temperature and time of the MIRA-LFD of the present invention, wherein: a is reaction temperature optimization, and b is reaction time optimization.
FIG. 4 is a primer-specific assay of the invention wherein: line1-7 is Eriocheir sinensis helicobacter, bacillus subtilis, bacillus thuringiensis, staphylococcus aureus, escherichia coli, aeromonas hydrophila, and Salmonella typhimurium respectively; and b, respectively preparing lines 1-8 from sterile water, bacillus subtilis, bacillus thuringiensis, staphylococcus aureus, escherichia coli, aeromonas hydrophila, salmonella and eriocheir sinensis spiroplasma.
Fig. 5 is a sensitivity test of the present invention, wherein: a is MIRA detection, b is MIRA-LFD detection, c is PCR detection, and the DNA concentration of the Eriocheir sinensis helicobacter used by Line1-9 is 10ng mu L respectively −1 、1 ng·μL −1 、100pg·μL −1 、10 pg·μL −1 、1 pg·μL −1 、100 fg·μL −1 、10 fg·μL −1 、1 fg·μL −1 Negative control.
FIG. 6 is a graph of the invention for repeated testing of clinical specimens using three methods, wherein: a is MIRA detection, b is MIRA-LFD detection, c is PCR detection; lines 1 and 2 in figures a and b are respectively negative control and positive control, and lines 3-19 are samples to be tested.
Description of the embodiments
The present invention is further illustrated in the following drawings and detailed description, which are to be understood as being merely illustrative of the invention and not limiting the scope of the invention.
Example 1: synthesis and screening of primers and probes
As shown in FIG. 1, according to the conservation sequence of the Eriocheir sinensis helicobacter, the MIRA primer design principle is combined: the primers of 30-35bp are used, no secondary structure exists, the amplified fragment size is 150-300bp, and 4 pairs of primers are designed through Olige6 software.
2 groups of primers with better amplification effect are selected from the 4 pairs of primers, and the design principle of the probe primers is adopted: in the middle of the upstream primer and the downstream primer, a sequence with the length of 46-52nt and complementary to the target fragment is designed, a FAM is modified at the 5' end, a dSpacer (tetrahydrofuran, THF) is marked at the middle position of the 5' end and the 3' ' end and is used as a recognition site of nfo, a modification group (C3 spacer) is marked at the 3' end, and 2 pairs of probe primers are designed through Olige6 software. The results showed that all primers amplified fragments of the expected size, the negative control did not show bands, and the positive control bands were bright and size-matched.
And finally screening the two groups of primers subjected to preliminary screening to finally obtain 1 group of 292F/446R primers with the fragment size of about 186bp and the expected size being consistent for detection by a follow-up MIRA method, and designing a group of probe primers for detection by the follow-up MIRA-LFD method by using the fragment.
Example 2: MIRA reaction system establishment
According to the specifications of the DNA isothermal rapid amplification kit (basic type) of the Biotechnology Co., ltd in the Datafang Anpu future, a 25. Mu.L MIRA reaction system was established, 29.4. Mu.L of a dissolution Buffer (A Buffer) was added to the lyophilized mixed enzyme, which was equally divided into 2 parts, followed by adding 1. Mu.L of Eriocheir sinensis helicobacter DNA per part, 6.05. Mu.L of sterile water, 1. Mu.L of 10. Mu. Mol/L forward primer, 1. Mu.L of 10. Mu. Mol/L reverse primer, and 1.25. Mu.L of magnesium acetate (B Buffer). The mixture is evenly mixed, vibrated for 1s, centrifuged for 1s, repeated twice and immediately placed into a water bath for reaction. MIRA product was purified by phenol: chloroform: isopropanol (25:24:1) followed by analysis by 2% agarose gel electrophoresis.
Example 3: MIRA-LFD reaction system establishment
According to the specifications of a DNA isothermal rapid amplification kit (colloidal gold test strip) of Datagram future biotechnology Co., ltd, a 25. Mu.L MIRA-LFD reaction system was established, 29.4. Mu.L of a dissolution Buffer (A Buffer) was added to the lyophilized mixed enzyme and divided into 2 parts, followed by 1. Mu.L of Eriocheir sinensis helicobacter DNA per part, 6.85. Mu.L of sterile water, 0.5. Mu.L of 1. Mu. Mol/L forward primer, 0.5. Mu.L of 1. Mu. Mol/L reverse primer, and 1.25. Mu.L of magnesium acetate (B Buffer). Mixing the mixture, shaking for 1s, centrifuging for 1s, repeating for two times, immediately placing into a water bath kettle for reaction, taking 10 mu L of reaction product after the reaction is finished, placing into a 20 mu L PCR small tube, adding 95 mu L of sterile enzyme-free water into the reaction product, mixing, inserting lateral chromatographic strips, standing for 5min, and observing the result.
The test strip result interpretation method comprises the following steps: when the control line (control line) is blue and the test line (test line) is red, the result is judged to be positive; the test strip quality control line is blue, and when the test line area is not provided with a strip, the result is judged to be negative; and when the test strip is not arranged in the quality control line and the test line area, the test strip is invalid, damaged or operated by mistake, and the detection result is invalid.
Example 4: condition optimization of MIRA, MIRA-LFD reaction system
(1) Reaction temperature optimization
As shown in FIGS. 2 and 3, the MIRA and MIRA-LFD reactions were performed at 25 ℃,30 ℃, 35 ℃, 37 ℃, 40 ℃ and 45 ℃ using the selected optimal MIRA and MIRA-LFD primers. After the reaction is finished, according to the detection results of agarose gel electrophoresis and a current-measuring chromatography test strip, the optimal reaction temperature of MIRA and MIRA-LFD is determined to be 37 ℃.
(2) Reaction time optimization
As shown in FIGS. 2 and 3, the selected optimal MIRA primers were used to react at 37℃for 1min,5min,10min,15min,20min,25min,30min, 35min, and 40min, respectively, and after the completion of the reaction, the detection results were determined by agarose gel electrophoresis. The selected MIRA-LFD primer is used for respectively reacting for 1min,5min,10min,15min,20min and 25min at 37 ℃, and the detection result is obtained by using a flow measurement chromatography test strip after the reaction is finished, and the result shows that the detection effect is optimal when the reaction time is 10min.
Example 5: specificity and sensitivity detection
The screened optimal primer and reaction condition are respectively used for detecting MIRA and MIRA-LFD by taking DNA of bacillus subtilis, bacillus thuringiensis, staphylococcus aureus, escherichia coli, aeromonas hydrophila, salmonella and eriocheir sinensis spiroplasma as templates and setting a blank control.
As shown in FIG. 4, the results show that the detection result has specific bands only when the DNA of the Eriocheir sinensis mycoplasma is used as a template, and no bands are generated in other samples, so that the established MIRA and MIRA-LFD detection methods of the Eriocheir sinensis mycoplasma have good specificity.
In addition, as shown in FIG. 5, the extracted samples DNA of Eriocheir sinensis helicobacter was diluted to 10 ng/. Mu.L respectively according to a 10-fold concentration gradient −1 、1 ng·μL −1 、100pg·μL −1 、10 pg·μL −1 、1 pg·μL −1 、100 fg·μL −1 、10 fg·μL −1 、1 fg·μL −1 A total of 8 different concentration gradients were measured using PCR, MIRA, MIRA-LFD. The detection result shows that the detection limit of the common PCR is 1 ng mu L −1 The method comprises the steps of carrying out a first treatment on the surface of the MIRA detection limit was 1 fg. Mu.L −1 The method comprises the steps of carrying out a first treatment on the surface of the MIRA-LFD detection limit is 1 fg mu L −1 The sensitivity of the MIRA and MIRA-LFD detection methods is the same and far higher than that of the common PCR.
In order to verify the feasibility of establishing the detection method, as shown in FIG. 6, 17 Eriocheir sinensis were randomly picked and subjected to MIRA, MIRA-LFD and PCR detection simultaneously, and the results indicate that 10 samples are positive, and the results of the three detection methods are consistent.
It should be noted that the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and it will be apparent to those skilled in the art that modifications and variations can be made in the above-described embodiment without departing from the principles of the present invention, and the modifications and variations fall within the scope of the appended claims.
Claims (8)
1. The detection method of the Eriocheir sinensis helicobacter MIRA and MIRA-LFD is characterized by comprising the following steps:
s1, synthesizing and screening a primer and a probe;
s2, establishing a MIRA reaction system;
s3, establishing a MIRA-LFD reaction system;
s4, specific detection.
2. The method for detecting the helicobacter Eriocheir sinensis MIRA and the MIRA-LFD according to claim 1, wherein the method comprises the following steps of: the specific operation steps of the step S1 are as follows:
(1) Designing a primer: using 30-35bp primer without secondary structure, amplifying fragment size 150-300bp, designing 4 pairs of primers by Olige6 software;
(2) Primary screening: designing a sequence with the length of 46-52nt and complementary to a target fragment in the middle of an upstream primer and a downstream primer, modifying a FAM at the 5 end, marking a dSpacer (tetrahydrofuran, THF) at the middle position of the 5 'end and the 3' 'end, marking a modification group (C3 spacer) at the 3' end as a recognition site of nfo, and designing 2 pairs of probe primers by Olige6 software;
(3) Final screening: and finally screening the two groups of primers to obtain 1 group of 292F/446R primers with the fragment size of about 186bp and the expected size being consistent for detection by a follow-up MIRA method, and designing a group of probe primers for detection by the follow-up MIRA-LFD method by using the fragment.
3. The method for detecting the helicobacter Eriocheir sinensis MIRA and the MIRA-LFD according to claim 1, wherein the method comprises the following steps of: the specific operation steps of the step S2 are as follows:
(1) 25. Mu.L of MIRA reaction system was established, 29.4. Mu.L of a dissolution Buffer (A Buffer) was added to the lyophilized mixed enzyme, and the mixture was divided into 2 portions;
(2) 1. Mu.L of Eriocheir sinensis helicobacter DNA, 6.05. Mu.L of sterile water, 1. Mu.L of 10. Mu. Mol/L forward primer, 1. Mu.L of 10. Mu. Mol/L reverse primer and 1.25. Mu.L of magnesium acetate (B Buffer) are added to each portion;
(3) Mixing the mixture uniformly, vibrating for 1s, centrifuging for 1s, repeating for two times, and immediately placing the mixture into a water bath kettle for reaction to obtain a MIRA product;
(4) The MIRA product was purified by phenol: chloroform: isopropanol=25: 24:1 followed by analysis by 2% agarose gel electrophoresis.
4. The method for detecting the helicobacter Eriocheir sinensis MIRA and the MIRA-LFD according to claim 1, wherein the method comprises the following steps of: the specific operation steps of the step S3 are as follows:
(1) Establishing a 25 mu L MIRA-LFD reaction system, adding 29.4 mu L dissolving Buffer (A Buffer) into the freeze-dried mixed enzyme, and dividing the mixture into 2 parts;
(2) 1. Mu.L of Eriocheir sinensis helicobacter DNA, 6.85. Mu.L of sterile water, 0.5. Mu.L of forward primer of 1. Mu.mol/L, 0.5. Mu.L of reverse primer of 1. Mu.mol/L, and 1.25. Mu.L of magnesium acetate (B Buffer) are added to each portion;
(3) Mixing the mixture uniformly, vibrating for 1s, centrifuging for 1s, repeating for two times, and immediately placing the mixture into a water bath kettle for reaction to obtain a MIRA-LFD product;
(4) 10. Mu.L of the reaction product was placed in a 20. Mu.L PCR cuvette, 95. Mu.L of sterile, sterile water was added thereto, mixed well, and a lateral chromatographic strip was inserted, allowed to stand for 5min, and the result was observed.
5. The method for detecting the helicobacter Eriocheir sinensis MIRA and the MIRA-LFD according to claim 1, wherein the method comprises the following steps of: the specific operation steps of the step S4 are as follows: the screened optimal primer and certain reaction conditions are used, and DNA of bacillus subtilis, bacillus thuringiensis, staphylococcus aureus, escherichia coli, aeromonas hydrophila, salmonella and eriocheir sinensis spiroplasma are used as templates, and a blank control is set at the same time to perform MIRA and MIRA-LFD detection.
6. The method for detecting the helicobacter Eriocheir sinensis MIRA and MIRA-LFD according to claim 5, wherein the method comprises the following steps of: the reaction conditions are specifically as follows: the reaction temperature of MIRA and MIRA-LFD is 37 ℃ and the reaction time is 10min.
7. Use of a detection method according to any one of claims 1 to 6 in a spiroplasma eri.
8. Use of the assay according to any one of claims 1-6 in the detection of clinical samples.
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