CN110396558B - Multiplex nested PCR (polymerase chain reaction) amplification primer and kit for simultaneously detecting five typical pathogens of cultured prawns - Google Patents
Multiplex nested PCR (polymerase chain reaction) amplification primer and kit for simultaneously detecting five typical pathogens of cultured prawns Download PDFInfo
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Abstract
The invention discloses a multiplex nested PCR amplification primer for simultaneously detecting five typical pathogens of cultured prawns, which comprises five pathogens SHIV, EHP, WSSV, IHHNV and VP AHPND Wherein: the invention also discloses a kit containing the primer and a non-disease detection target multiplex PCR detection method for detecting five pathogens by adopting the primer, and parameters such as annealing temperature, dNTP, EX Taq enzyme, PCR reaction concentration of five prawn pathogen primers and the like of external amplification and internal amplification reactions are optimized and determined by designing external amplification and internal amplification primers, so that the requirements of simultaneously detecting SHIV, EHP, WSSV, IHHNV and VP with larger production hazard of prawn culture are met on one hand AHPND The requirements of five pathogens are met, and on the other hand, higher sensitivity can be achieved.
Description
Technical Field
The invention belongs to the technical field of prawn pathogen detection, and particularly relates to a multiplex nested PCR (polymerase chain reaction) amplification primer and a kit for simultaneously detecting five typical pathogens of cultured prawns, and more particularly relates to a multiplex nested PCR amplification primer, a non-disease detection target multiplex nested PCR detection method for simultaneously detecting the five typical pathogens by adopting the primer and application of the multiplex nested PCR amplification primer.
Background
Litopenaeus vannamei (Litopenaeus vannamei), also known as Penaeus vannamei, is native to the coastal areas of Pacific America, and is popular with the public because of its advantages of wide adaptability to salinity, fast growth, delicious meat, low nutritional requirements, and strong adaptability. According to the statistics of Chinese fishery, the total yield of the penaeus vannamei boone in 2017 accounts for more than 75% of the total yield of the penaeus vannamei boone in China. Although the economic benefit and annual yield of the penaeus vannamei boone generally increase, the penaeus vannamei boone cultivation yield in 2015-2017 is a slow-growing stage, and the industrial development encounters a bottleneck. One of the important reasons is that the disease influence is serious, once small-scale diseases occur in the cultivation production process, the diseases can directly cause explosive spread if the treatment is not properly controlled, and huge losses are caused. According to Zheng Xiaoshe and other researches, common pathogens are not optimistic for the infection condition of shrimp fries in main culture areas of the penaeus vannamei boone in China, and most of the easily-perceived diseases of the penaeus vannamei boone do not have effective cure means. For this, detailed knowledge of common pathogens is necessary to grasp the dynamics of the culture pond in real time, minimizing losses. According to the research on pathogens in different regions of China in 2013-2018 of Liang Jingzhen, wang Boya, wu Heying and Zhang Na, the disease types and epidemic conditions in different regions are different, and mainly comprise shrimp blood cell iridovirus (Shrimp hemocyte iridescent virus, SHIV), shrimp liver and intestinal cyst (Enterocytozoon hepatopenaei, EHP) and halophilic Vibrio parahaemolyticus (Vibrio parahaemolyticusAHPND, VP) AHPND ) White spot syndrome virus (White spot syndrome virus, WSSV), infectious hypodermia and its productionFive pathogens, blood tissue necrosis virus (Infectious hypodermal and hernatopoietic necrosis virus, IHHNV), are the major. The positive rate of the WSSV of the litopenaeus vannamei in the coastal areas of Guangxi in 2015 and 2016 is 30.4 percent and 41.9 percent respectively; wang Boya (2017) and the like have an IHHNV positive rate of 65.4% and an EHP positive rate of 34.6% detected in 26 prawn disease ponds in Liaoning province. The detection of EHP and VP in the culture pond of almost all follow-up surveys was found in the follow-up surveys of the pathogens of the farms by Wu and Ying et al in 2017 AHPND . The investigation of 2017, 4-6 months Wang Ganxiang and the like in Pinghu city of Zhejiang province shows that the phenomenon of death of the prawns in the culture pond caused by SHIV infection occurs in the city.
Therefore, the pathogen detection of the prawns is carried out early, the pathogen carrying conditions of the young prawns, the culture water body, the input products and the like are mastered, and the method has great significance for the culture production of the prawns. The existing prawn pathogen detection technology method mainly comprises histopathological diagnosis, colloidal gold immunochromatography detection method, molecular biology method and the like. The molecular biological method has the advantages of simplicity, convenience, high sensitivity and the like, and is widely applied to the detection of the prawn pathogen. In the aspect of research and development of related pathogen detection technology, yellow sea aquatic products research institute yellow team of Chinese aquatic products science institute developed a nest type PCR detection technology for SHIV pathogen, a fluorescent quantitative PCR detection technology for EHP and IHHNV pathogen and a TaqMan probe fluorescent quantitative PCR detection technology, and a VP detection technology AHPND Fluorescent quantitative PCR detection technology of pathogens; team He Jianguo of university of Zhongshan developed PCR detection technology, in situ hybridization detection technology and nested PCR detection technology for WSSV pathogens; group Yang Feng of the institute of Nature resources, third sea developed an antiviral agent against the red swamp crayfish iridovirus (Cherax quadricarinatus iridovirus, CQIV), EHP and VP AHPND Fluorescent quantitative PCR detection technology of isopathogenic substances and multiplex fluorescent quantitative PCR detection technology thereof.
Among them, multiplex nested PCR has the advantage in practical application: compared with single PCR, multiple nested PCR can detect multiple pathogens at one time, the detection is rapid, the period is short, and the consumed manpower and material resources are less than those of the single PCR; compared with multiple common PCRs, the multiple nested PCRs are amplified twice, so that the mismatch probability is reduced, and the detection reliability and sensitivity are improved; compared with fluorescent quantitative PCR and gene chip technology, the multiplex nested PCR has lower experiment cost, does not need expensive precise instruments and reagents, and can observe the size of a target fragment; compared with the LAMP technology, the multiplex nested PCR primer has lower design requirement, lower probability of false positive and nonspecific amplification and wider range of detectable target nucleic acid.
But aiming at SHIV, EHP, WSSV, IHHNV and VP which have great harm to the prawn breeding industry in recent years AHPND Multiple nested PCR detection techniques for five typical pathogens are reported.
Disclosure of Invention
The invention aims to provide a multiplex nested PCR amplification primer for simultaneously detecting five typical pathogens of cultured prawns.
The invention also aims at providing a multiplex nested PCR detection kit for simultaneously detecting five typical pathogens of the cultured prawns.
The third object of the invention is to provide a multiplex nested PCR detection method for detecting five typical pathogens of cultured prawns by using the primer.
The final object of the invention is to provide the application of the primer and the kit in the detection of five typical pathogenic non-disease detection purposes of the cultured prawns simultaneously.
The first object of the present invention can be achieved by the following technical scheme: a multiplex nested PCR amplification primer for simultaneously detecting five typical pathogens of cultured prawns comprises five pathogens SHIV, EHP, WSSV, IHHNV and VP AHPND Wherein:
the sequence of the external amplification primer of the pathogenic SHIV is shown as SEQ ID NO. 1-2;
the internal amplification primer sequence of the pathogenic SHIV is shown as SEQ ID NO. 3-4;
the sequence of the external amplification primer of the pathogenic EHP is shown as SEQ ID NO. 5-6;
the internal amplification primer sequence of the pathogenic EHP is shown in SEQ ID NO. 7-8;
the sequence of the external amplification primer of the pathogenic WSSV is shown as SEQ ID NO. 9-10;
the internal amplification primer sequence of the pathogen WSSV is shown as SEQ ID NO. 11-12;
the sequence of the external amplification primer of the pathogenic IHHNV is shown in SEQ ID NO. 13-14;
the internal amplification primer sequence of the pathogenic IHHNV is shown in SEQ ID NO. 15-16;
the pathogenic VP AHPND The sequence of the outer amplification primer of the primer is shown as SEQ ID NO. 17-18;
the pathogenic VP AHPND The internal amplification primer sequences of (2) are shown as SEQ ID NO. 19-20.
The invention firstly screens out the prawn target pathogenic prawn blood cell iridovirus (Shrimp hemocyte iridescent virus, SHIV), prawn liver and intestine cytozoon (Enterocytozoon hepatopenaei, EHP) and halophilic vibrio parahaemolyticus (Vibrio parahaemolyticus AHPND, VP) AHPND ) As a detection index; then, the white spot syndrome virus (White spot syndrome virus, WSSV) and infectious subcutaneous and hematopoietic necrosis virus (Infectious hypodermal and hernatopoietic necrosis virus, IHHNV) which are emphasized by OIE are selected into a detection index system. Final establishment of Simultaneous detection SHIV, EHP, WSSV, IHHNV and VP AHPND And five kinds of multiplex nested PCR primers for prawn pathogens.
The invention obtains SHIV, EHP, WSSV, IHHNV and VP from GenBank AHPND The DNA sequences of five pathogenic genes were alike, for which specific nested PCR inner and outer primers were designed respectively using Primer 5 design analysis software, for a total of 10 pairs (see Table 1 below). Meanwhile, DNAstar analysis software is used for analyzing the sequences of the PCR primers to prevent the problem that the homology and complementarity between the primers of each group are too high and dimers are formed between the primers.
TABLE 1 primers for multiplex nested PCR technical method for simultaneously detecting five typical prawn pathogens developed by the invention
The second object of the present invention can be achieved byThe method is realized by the following technical scheme: a multiplex nested PCR detection kit for simultaneously detecting five typical pathogens of cultured prawns comprises an external amplification reaction solution, an internal amplification reaction solution, a positive control and a negative control; wherein the external amplification reaction solution comprises the external amplification primer, dNTP, EXTaq enzyme and ddH 2 O, the internal amplification reaction solution comprises the internal amplification primer, dNTP, EXTaq enzyme and ddH 2 O。
The multiple nested PCR detection method aiming at the five typical pathogens is not just a simple combination of single pathogen detection methods, and each condition parameter of the multiple nested PCR reaction system needs to be optimized and improved so as to avoid the inhibition effect of each condition in the reaction system.
Based on the specific requirements of the multiplex nested PCR detection of the five typical pathogens, the invention optimally designs the external amplification primer and the internal amplification primer, and tests and determines the PCR reaction concentration parameters of dNTPs, EX Taq enzyme and the five typical pathogens.
Preferably, the external expansion reaction solution is 25 μl of a reaction system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the primers used for the upstream and downstream amplification of the five typical pathogens was 0.32. Mu. Mol/L.
Preferably, the internal amplification reaction solution is 25. Mu.L of a reaction system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the inner amplification upstream and downstream primers of the five typical pathogens is 0.48 mu mol/L.
The third object of the present invention can be achieved by the following means: the multiplex nested PCR detection method for detecting five typical pathogens of cultured prawns by using the primer comprises the following steps:
(1) Constructing a positive control;
(2) Extracting DNA of a sample to be detected;
(3) And (3) performing an external amplification PCR reaction: constructing an external amplification PCR reaction system and an external amplification PCR reaction program, and performing an external amplification PCR reaction to obtain an external amplification product;
(4) Internal amplification PCR reaction: constructing an internal amplification PCR reaction system and an internal amplification PCR reaction program, and carrying out an internal amplification PCR reaction on the diluted external amplification product to obtain an internal amplification product;
(5) And (3) judging a PCR result: and detecting an internal amplification product by adopting electrophoresis, wherein if a positive strip with the same size as the positive control fragment appears, the biological sample carries a corresponding target pathogen.
The multiple nested PCR detection method aiming at the five typical pathogens is not just a simple combination of single pathogen detection methods, and each condition parameter of the multiple nested PCR reaction system needs to be optimized and improved so as to avoid the inhibition effect of each condition in the reaction system.
Based on the specific requirements of the multiplex nested PCR detection of the five typical pathogens, the invention optimally designs the external amplification primer and the internal amplification primer, and tests and determines the annealing temperature, dNTP, EX Taq enzyme and the PCR reaction concentration parameters of the five pathogen primers of the external amplification reaction system and the internal amplification reaction system.
Therefore, in the multiplex nested PCR detection method for detecting five typical pathogens of the cultured prawns by adopting the primer, the detection method comprises the following steps:
preferably, the step (3) of the external expansion reaction is a 25. Mu.L system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the external amplification upstream and downstream primers of the five pathogens is 0.32 mu mol/L.
Preferably, the step (3) comprises the following steps: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 61℃for 1min, extension at 72℃for 1min, and 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
Preferably, the internal amplification reaction described in step (4) is a 25. Mu.L system wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the inner amplification upstream and downstream primers of the five pathogens is 0.48 mu mol/L.
Preferably, the internal amplification reaction procedure in step (4) is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, and 35 cycles were performed; finally, the extension is carried out for 10min at 72 ℃.
Specific: the optimal reaction system for the external amplification and internal amplification reaction of the multiplex nested PCR detection method for simultaneously detecting five shrimp pathogens, which is established by the invention, is shown as follows.
The optimal reaction system for the multiplex nested PCR detection method external amplification reaction is shown in Table 2.
The external amplification reaction program of the multiplex nested PCR detection method is that the multiplex nested PCR detection method is pre-denatured for 3min at 95 ℃; denaturation at 95℃for 30s, annealing at 61℃for 1min, extension at 72℃for 1min, and 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
Table 2 multiple nest PCR detection technique method external expansion reaction system
The optimal reaction system for the internal amplification reaction of the multiplex nested PCR detection method is shown in Table 3.
The internal amplification reaction program of the multiplex nested PCR detection technical method is pre-denatured for 3min at 95 ℃; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, and 35 cycles were performed; finally, the extension is carried out for 10min at 72 ℃.
Table 3 internal amplification reaction system of multiplex nest type PCR detection technical method
The multiplex nested PCR method pair SHIV, EHP, WSSV, IHHNV and VP AHPND The PCR amplified fragments of (2) have the sizes of 129bp, 176bp, 230bp, 294bp and 402bp respectively. Wherein the optimal annealing temperature for the external amplification is 61 ℃, the optimal concentration of the external primer is 0.32 mu mol/L, and the optimal concentrations of EX Taq and dNTP are 1.5U/25 mu L and 1.5mmol/L respectively; the optimal annealing temperature for internal amplification is 60 ℃, the optimal concentration of the internal primer is 0.48 mu mol/L, and the optimal concentrations of EX Taq and dNTP are 1.5U/25 mu L and 1.5mmol/L respectively.
The final object of the invention can be achieved by the following technical scheme: the primer and the kit are applied to the detection of five typical pathogenic non-diseases of the cultured prawns simultaneously.
By using the primer or the kit of the inventionThe detection is that the minimum detection amount of IHHNV by the multiplex nested PCR detection method is 1 multiplied by 10 2 COPies/. Mu.L for VP AHPND The lowest detection amount of WSSV, EHP, SHIV is 1×10 1 COPies/. Mu.L. The applicability of the method is verified by samples such as shrimp seedlings, adult shrimps, artemia cysts and the like of the litopenaeus vannamei collected from Guangdong Shandong tail and flourishing main production areas.
Compared with the prior art, the invention has the following advantages:
(1) The invention aims at five typical pathogens which have great harm to the prawn breeding industry in recent years, namely, prawn blood cell iridovirus (SHIV), prawn liver and intestine cyst (EHP), white Spot Syndrome Virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and halophilic Vibrio Parahaemolyticus (VP) AHPND ) Designing external amplification and internal amplification primers, optimizing and determining parameters such as annealing temperature, dNTP, EX Taq enzyme, PCR reaction concentration of five prawn pathogenic primers and the like of external amplification and internal amplification reactions. On one hand, the method can be ensured to simultaneously detect SHIV, EHP, WSSV, IHHNV and VP with larger harm in the shrimp culture production AHPND The requirements of five pathogens are equal; on the other hand, the method of the invention can reach higher sensitivity, and the lowest detection amount of IHHNV is 1 multiplied by 10 2 COPies/. Mu.L for VP AHPND The lowest detection amount of WSSV, EHP, SHIV is 1×10 1 copies/μL。
(2) The method has good application prospect and can provide technical support for the subsequent further optimization and integrated shrimp disease ecological prevention and control technical system.
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FIG. 1 is a diagram showing the detection results of multiplex nested PCR sensitivity in example 2, wherein M is 100bp DNA Ladder; lanes 1-6 are 1X 10, respectively 6 copies/μL，1×10 5 copies/μL，1×10 4 copies/μL，1×10 3 copies/μL，1×10 2 copies/μL，1×10 1 Five pathogenic positive plasmids of copies/. Mu.L;
FIG. 2 is the result of multiplex nested PCR detection of five pathogens in shrimp larvae samples of example 3, M being 100bp DNA Ladder; PC is positive control; NC is negative control; 1-15 is a detected shrimp seed sample;
FIG. 3 is the result of multiplex nested PCR detection of five pathogens in a artemia cysts sample of example 3, M being 100bp DNA Ladder; PC is positive control; NC is negative control; 16-30 are the detected artemia cysts;
FIG. 4 is the result of multiplex nested PCR detection of five pathogens of a portion of the sample of cultured prawns in example 3, M being 100bp DNA Ladder; PC is positive control; NC is negative control; 31 to 45 are samples of the adult shrimps tested.
Detailed Description
The application method of the present invention will be further described with reference to the following specific examples. The following examples and figures are for illustrative purposes only and are not to be construed as limiting the invention. Unless otherwise indicated, the reagent raw materials used in the following examples are raw reagent raw materials which are conventionally commercially available or commercially available, and the methods and apparatuses used in the following examples are methods and apparatuses conventionally used in the art unless otherwise indicated.
EXAMPLE 1 specific primer design for five prawn pathogens
The invention firstly screens out the prawn target pathogenic prawn blood cell iridovirus (Shrimp hemocyte iridescent virus, SHIV), prawn liver and intestine cytozoon (Enterocytozoon hepatopenaei, EHP) and halophilic vibrio parahaemolyticus (Vibrio parahaemolyticus AHPND, VP) AHPND ) As a detection index; then, the white spot syndrome virus (White spot syndrome virus, WSSV) and infectious subcutaneous and hematopoietic necrosis virus (Infectious hypodermal and hernatopoietic necrosis virus, IHHNV) which are emphasized by OIE are selected into a detection index system. Final establishment of Simultaneous detection SHIV, EHP, WSSV, IHHNV and VP AHPND And five kinds of multiplex nested PCR primers for prawn pathogens.
SHIV, EHP, WSSV, IHHNV and VP available from GenBank AHPND The DNA sequences of five pathogenic genes were alike, and specific nested PCR inner and outer primers were designed for them by Primer 5 design analysis software, for a total of 10 pairs (see Table 1). At the same time, the PCR primer sequences were analyzed using DNAstar analysis software to prevent each set of primersToo high homology and complementarity, and the formation of dimers between the primers.
TABLE 1 primers for multiplex nested PCR technical method for simultaneously detecting five typical prawn pathogens developed by the present invention
Specifically, the multiplex nested PCR amplification primer comprises five pathogens SHIV, EHP, WSSV, IHHNV and VP AHPND Wherein:
the sequence of the external amplification primer of the pathogenic SHIV is shown as SEQ ID NO. 1-2;
the internal amplification primer sequence of the pathogenic SHIV is shown as SEQ ID NO. 3-4;
the sequence of the external amplification primer of the pathogenic EHP is shown as SEQ ID NO. 5-6;
the internal amplification primer sequence of the pathogenic EHP is shown in SEQ ID NO. 7-8;
the sequence of the external amplification primer of the pathogenic WSSV is shown as SEQ ID NO. 9-10;
the internal amplification primer sequence of the pathogen WSSV is shown as SEQ ID NO. 11-12;
the sequence of the external amplification primer of the pathogenic IHHNV is shown in SEQ ID NO. 13-14;
the internal amplification primer sequence of the pathogenic IHHNV is shown in SEQ ID NO. 15-16;
the pathogenic VP AHPND The sequence of the outer amplification primer of the primer is shown as SEQ ID NO. 17-18;
the pathogenic VP AHPND The internal amplification primer sequences of (2) are shown as SEQ ID NO. 19-20.
The primer can be used for preparing a multiplex nested PCR detection kit for simultaneously detecting five typical pathogens of the cultured prawns.
Preferably, the method is used for simultaneously detecting five typical pathogens of the cultured prawnsThe multiplex nested PCR detection kit comprises an external amplification reaction solution, an internal amplification reaction solution, a positive control and a negative control; wherein the external amplification reaction solution comprises the external amplification primer, dNTP, EXTaq enzyme and ddH 2 O, the internal amplification reaction solution comprises the internal amplification primer, dNTP, EXTaq enzyme and ddH 2 O。
Preferably, in the external expansion reaction solution: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U/25. Mu.L, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the external amplification upstream and downstream primers of the five typical pathogens is 0.32 mu mol/L; in the internal amplification reaction, dNTP concentration is 1.5mmol/L, EX Taq enzyme 1.5U/25 μL, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the inner amplification upstream and downstream primers of the five typical pathogens is 0.48 mu mol/L.
Example 2 non-disease detection target multiplex nested PCR detection method for five pathogens using the above primers
(1) Preparation of five positive plasmids for prawn pathogen
1.1 screening of prawn pathogenic Positive amplicons
Collecting diseased prawns with the five pathogens as positive samples, extracting DNA as templates, and carrying out conventional PCR amplification by using the primers designed as above.
The reaction system was 15. Mu.L, including 1. Mu.L of DNA template of the shrimp sample, 7.5. Mu.L of Premix TaqTM mix (TaKaRa), 0.5. Mu.L of upstream primer (10. Mu.M), 0.5. Mu.L of downstream primer (10. Mu.M), and 5.5. Mu.L of ddH2O.
The PCR amplification procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 60℃for 60s, extension at 72℃for 60s, 30 cycles; finally, the extension is carried out for 10min at 72 ℃.
1.2 recovery of prawn pathogen Positive amplicon DNA cutting gel
The PCR products were subjected to 1% agarose gel electrophoresis, and the electrophoresis results were observed under a gel imager. The target amplified bands of the five pathogenic genes are respectively cut in a Lan Guangqie glue instrument,
according to the operation steps of the agarose gel DNA recovery kit, the positive amplicon DNA of the prawn pathogenic gene is recovered and purified. The purified DNA was stored at-80℃for later use.
1.3 vector ligation of shrimp pathogen Positive amplicon DNA
The purified prawn pathogenic positive amplicon DNA is connected with pEASY-T1 cloning vector, and the specific connection reaction system comprises pEASY-T1 Simple Cloning Vector mu L and 4 mu L of prawn pathogenic positive amplicon DNA. The EP tube containing the connection reaction system was gently mixed and allowed to stand at 25℃for 5min. After the reaction was completed, the EP tube was placed on ice.
1.4 preparation of a Standard Strain of a prawn pathogen Positive plasmid
The above-described product of the pEASY-T1 cloning vector was added to 50. Mu.L of trans1-T1 competent cells, plasmid transformation competent cells were completed according to the procedure of the pEASY cloning vector specification, and then spread on an agar plate containing IPTG and X-gal, and cultured in an incubator at 37℃overnight with inversion. White clones were selected and inoculated into LB/Amp+ liquid medium, cultured at 200rpm and 37℃overnight, and plasmid DNA was extracted using plasmid miniprep kit. Amplifying the obtained pathogenic plasmid DNA by using each prawn pathogenic DNA primer according to the reaction procedure, carrying out 1.5% agarose gel electrophoresis detection positive sequencing on the PCR product, and comparing and confirming the DNA which is the target pathogen on NCBI.
1.5 calculation of the concentration of the shrimp pathogen Positive plasmid
The DNA concentration of the prawn pathogenic positive plasmid is determined by NanoDrop 2000 after sequencing and comparison, and the specific volume pathogenic copy number (copies/mu L) of the prawn pathogenic positive plasmid is calculated by combining the DNA sequence of each pathogenic positive amplicon and the pEASY-T1 cloning vector sequence, and diluted to 1X 10 according to 10 times gradient 1 The copies/. Mu.L was stored at-80℃until use.
(2) Multiple nest type PCR reaction condition optimization-multiple nest type PCR external expansion reaction condition optimization of five kinds of prawn pathogens
Optimization of optimal primer concentration in the external amplification reaction: the concentration of the five plasmids was 3X 10 using 25. Mu.L of the amplification system 7 Copy/. Mu.L, cycle times were set to 35 times, annealing temperature was set to 60℃and Premix Taq TM (TaKaRa) was set to 1.5U/25. Mu.L, dNTP concentration was set to 2.0mmol/L, and the concentration gradient of the upstream and downstream primers was set to 0.16. Mu.LPCR amplification was performed at 0.32. Mu. Mol/L, 0.48. Mu. Mol/L, 0.64. Mu. Mol/L, and 0.80. Mu. Mol/L, and multiplex nested PCR optimal outer primer concentrations were searched.
Optimization of optimal dNTP concentration in the external amplification reaction: adopting a 25 mu L amplification system, adopting the optimal primer concentration obtained in the last step for the concentration of the upstream primer and the downstream primer, setting the concentration gradient of dNTPs to be 1.0mmol/L, 1.5mmol/L, 2.0mmol/L and 2.5mmol/L, and searching the optimal DNTP concentration of the multiplex nested PCR under the condition of unchanged other reaction conditions.
Optimization of optimal enzyme concentration in the external amplification reaction: the concentration gradient of EX Taq enzyme was set to 1.0U/25. Mu.L, 1.5U/25. Mu.L, 2.0U/25. Mu.L, 2.5U/25. Mu.L, using the optimal outer primer and dNTP concentrations previously obtained, with the other reaction conditions unchanged.
Optimization of the optimal annealing temperature in the external expansion reaction: the optimal annealing temperature gradient is set to 58 ℃, 59 ℃,60 ℃,61 ℃ by adopting the optimal outer primer, EX Taq enzyme and dNTP concentration obtained before, and other reaction conditions are unchanged, so as to search the optimal annealing temperature of the multiplex nested PCR.
As a result, the primer concentration in the external amplification reaction was 0.32. Mu. Mol/L, dNTP at 1.5mmol/L, the enzyme concentration was 1.5U/25. Mu.L, and the 5-piece bands 457bp, 545bp, 700bp, 802bp and 925bp were uniform and clear in brightness at 61 ℃.
(3) Multiple nest type PCR reaction condition optimization-multiple nest type PCR internal amplification reaction condition optimization of five prawn pathogens
Optimal inner primer concentration optimization in the inner amplification reaction: the concentration of the five plasmids was 3X 10 using 25. Mu.L of the amplification system 7 The cycles were set to 35 times, the annealing temperature was set to 60℃and the Premix Taq TM (EX TaKaRa) was set to 2.0U/25. Mu.L, dNTP concentration was set to 2.5mmol/L, and the concentration gradients of the upstream and downstream primers were set to 0.48. Mu. Mol/L, 0.64. Mu. Mol/L, 0.80. Mu. Mol/L, and 0.96. Mu. Mol/L for PCR amplification, and multiplex nested PCR was searched for optimal inner primer concentrations.
Optimizing the optimal dNTP concentration in the internal amplification reaction: adopting a 25 mu L amplification system, adopting the optimal primer concentration obtained in the last step for the concentration of the upstream primer and the downstream primer, setting the concentration gradient of dNTPs to be 1.5mmol/L, 2.0mmol/L, 2.5mmol/L and 3.0mmol/L, and searching the optimal dNTP concentration of the multiplex nested PCR under the condition of unchanged other reaction conditions.
Optimizing the optimal enzyme concentration in the internal amplification reaction: the concentration gradient of EX Taq enzyme was set to 1.0U/25. Mu.L, 1.5U/25. Mu.L, 2.0U/25. Mu.L, 2.5U/25. Mu.L, using the optimal inner primer and dNTP concentrations previously obtained, with other reaction conditions unchanged, and multiplex nested PCR optimal enzyme concentrations were searched.
Optimizing the optimal annealing temperature in the internal amplification reaction: the optimal annealing temperature gradient is set to 59 ℃,60 ℃,61 ℃, 62 ℃ by adopting the optimal inner primer, EX Taq enzyme and dNTP concentration obtained before, and other reaction conditions are unchanged, so as to search the optimal annealing temperature of the multiplex nested PCR.
As a result, the primer concentration in the internal amplification reaction was 0.48. Mu. Mol/L, dNTP and 1.5mmol/L, the enzyme concentration was 1.5U/25. Mu.L, and the annealing temperature was 60℃and the 5-piece bands were 129bp, 176bp, 230bp, 294bp, and 402bp, which were uniform and clear in brightness.
(4) Sensitivity detection of five multi-nest PCR detection methods for prawn pathogens
SHIV, EHP, VP the above-mentioned AHPND Positive plasmids of five pathogens, such as WSSV, IHHNV and the like, are 1 multiplied by 10 1 ～1×10 6 The concentration of copies/. Mu.L was diluted in a 10-fold gradient. Respectively 1×10 1 ～1×10 6 The plasmid with the concentration is used as a template, and the optimized five-nest PCR detection method is adopted for amplification so as to detect the sensitivity.
As a result, as shown in FIG. 1, it can be seen from FIG. 1 that 5 amplified bands of the target pathogen appear in lanes 1 to 5, 4 amplified bands of the target pathogen appear in lane 6, and IHHNV pathogen amplified band at 294bp position does not appear. The result shows that the minimum detection concentration of IHHNV by the multiple nest PCR detection method of five prawn pathogens is 1 multiplied by 10 2 COPIES/. Mu.L, for the remaining 4 pathogenic VP AHPND The lowest detection concentration of the primer and WSSV, EHP, SHIV can reach 1 multiplied by 10 1 The copies/. Mu.L has higher detection sensitivity.
Example 3 use of prawn pathogen detection
Samples such as litopenaeus vannamei larvae, adult shrimps, artemia cysts and the like are collected in shrimp culture production bases of Guangdong tail American village and Maoque electric white-mountain men town in 2017 to 2019 respectively. The method is used for detecting and applying the five shrimp pathogens in the laboratory of the south China aquatic institute.
First, biological tissue DNA is extracted. About 30mg of the hepatopancreatic tissue of the prawn, the whole individual was sampled from the young prawn, about 30mg of the sample was sampled from the artemia egg, and the biological tissue DNA was extracted according to the E.Z.N.A.tissue DNAKit kit instruction of Omega company in the United states.
Five typical prawn pathogens were then detected on the extracted biological tissue DNA using the primers of example 1 and the multiplex nested PCR detection method of example 2.
Firstly, carrying out pathogenic external amplification PCR reaction, constructing 25 mu L of external amplification PCR system, wherein dNTP concentration is 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the external amplification upstream and downstream primers of the five pathogens is 0.32 mu mol/L.
The external expansion reaction procedure is that the pre-denaturation is carried out for 3min at 95 ℃; denaturation at 95℃for 30s, annealing at 61℃for 1min, extension at 72℃for 1min, and 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
Positive plasmid DNA and sterile water for each pathogen served as positive and negative controls, respectively.
After the outer-amplification PCR reaction is completed, the outer-amplification PCR product is diluted 10 times and used as a template for the inner-amplification PCR reaction.
Construction of a 25. Mu.L internal amplification PCR System with dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the inner amplification upstream and downstream primers of the five pathogens is 0.48 mu mol/L.
The internal amplification reaction procedure is that the pre-denaturation is carried out for 3min at 95 ℃; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, and 35 cycles were performed; finally, the extension is carried out for 10min at 72 ℃.
And after the internal amplification PCR reaction is finished, detecting the internal amplification PCR amplification condition by adopting 2% agarose gel electrophoresis, and analyzing and photographing by using a gel imager system after electrophoresis.
The detection results are shown in FIGS. 2 to 4 below, and SHIV (1) is obtained in the positive control lane (PC)29 bp), EHP (176 bp), WSSV (230 bp), IHHNV (294 bp) and VP AHPND (402 bp) target amplified fragments of five pathogens. The negative control lane (NC) had no amplified fragment bands. Lanes 1-15 are the conditions of the target pathogen carried by the sample being detected, and in some of the detected samples, positive bands consistent with the positive control amplified fragments appear, so that the sample carries the corresponding target pathogen.
FIGS. 2, 3 and 4 show SHIV (129 bp), EHP (176 bp), WSSV (230 bp), IHHNV (294 bp) and VP using the method of the present invention in the presence of a Positive Control (PC) and a Negative Control (NC) AHPND (402 bp) five pathogens were multiplex-nested PCR amplified and the PCR amplified products were subjected to 2% agarose gel electrophoresis. The Positive Control (PC) of the three figures all have the amplified bands of the 5 target pathogens, and the Negative Control (NC) has no band, so that the method has good quality control, is feasible and can detect the target pathogens.
The result of FIG. 2 is that the amplified bands of samples 2, 5, 11 are identical to the 176bp positional band of the EHP pathogen in the positive control, indicating that the samples carry the EHP pathogen.
The result of FIG. 3 is that the amplified bands of samples 16, 18, 19, 21, 22, 24, 25, 26, 27, 28, 29 are identical to the 129bp positional band of the SHIV pathogen in the positive control, indicating that the samples carry the SHIV pathogen; the amplified bands of samples 16, 17, 18, 23, 26 were identical to the 176bp positional band of EHP pathogen in the positive control, indicating that the samples carry EHP pathogen.
The result of FIG. 4 is that the amplified bands of samples 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 are identical to the 129bp position band of SHIV pathogen in the positive control, indicating that the samples carry SHIV pathogen; the amplified bands of samples 35, 37, 38, 40, 41, 44 were identical to the 176bp positional band of EHP pathogen in the positive control, indicating that the samples carried EHP pathogen; the amplified bands of samples 35, 38, 41, 44 were identical to the 230bp position band of the WSSV pathogen in the positive control, indicating that the samples carried the WSSV pathogen.
The detection result shows that the primer and the method can simultaneously detect SHIV, EHP, WSSV, IHHNV and VP in the sample AHPND Five pathogens are detected in a large quantity and accurately and conveniently, and the method has good industrial application prospect.
The invention is not limited to the specific embodiments described above, which are only intended to be able to describe in detail the course of use of the invention, and also the production methods and technical details with equivalent functions are part of the present disclosure. Indeed, those skilled in the art will be able to find different adjustment schemes according to the needs of each, and these adjustments are within the scope of the claims appended hereto.
Sequence listing
<110> institute of aquatic products in south China national institute of aquatic products
<120> a multiplex nested PCR amplification primer and kit for simultaneously detecting five typical pathogens of cultured prawns
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<211> 21
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<213> shrimp blood cell iridovirus (Shrimp hemocyte iridescent virus)
<400> 2
tcgtttcggt acgaagatgt a 21
<210> 3
<211> 21
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<213> shrimp blood cell iridovirus (Shrimp hemocyte iridescent virus)
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<213> shrimp blood cell iridovirus (Shrimp hemocyte iridescent virus)
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<213> shrimp liver and intestine cyst insect (Enterocytozoon hepatopenaei)
<400> 6
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<213> shrimp liver and intestine cyst insect (Enterocytozoon hepatopenaei)
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Claims (9)
1. A multiplex nested PCR amplification primer group for simultaneously detecting five typical pathogens of cultured prawns is characterized by comprising five pathogens SHIV, EHP, WSSV, IHHNV and VP AHPND Wherein:
the sequence of the external amplification primer of the pathogenic SHIV is shown in SEQ ID NO. 1-2;
the internal amplification primer sequence of the pathogenic SHIV is shown in SEQ ID NO. 3-4;
the sequence of the external amplification primer of the pathogenic EHP is shown in SEQ ID NO. 5-6;
the internal amplification primer sequence of the pathogenic EHP is shown in SEQ ID NO. 7-8;
the sequence of the external amplification primer of the pathogenic WSSV is shown in SEQ ID NO. 9-10;
the internal amplification primer sequence of the pathogenic WSSV is shown in SEQ ID NO. 11-12;
the sequence of the external amplification primer of the pathogenic IHHNV is shown in SEQ ID NO. 13-14;
the internal amplification primer sequence of the pathogenic IHHNV is shown in SEQ ID NO. 15-16;
the pathogen V PAHPND The sequence of the outer amplification primer of (2) is shown as SEQ ID NO. 17-18;
the pathogenic VP AHPND The internal amplification primer sequence of (2) is shown as SEQ ID NO. 19-20.
2. A multiplex nested PCR detection kit for simultaneously detecting five typical pathogens of cultured prawns is characterized in that: comprises an external amplification reaction solution, an internal amplification reaction solution, a positive control and a negative control; wherein the reaction solution for external amplification comprises the external amplification primer, dNTP, EX Taq enzyme and ddH according to claim 1 2 O, the internal amplification reaction solution comprising the internal amplification primer, dNTP, EXTaq enzyme and ddH of claim 1 2 O。
3. A kit according to claim 2, characterized in that: the external expansion reaction liquid is a 25 mu L reaction system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the external amplification upstream and downstream primers of the five typical pathogens is 0.32 mu mol/L; the internal amplification reaction solution is a 25 mu L reaction system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the inner amplification upstream and downstream primers of the five typical pathogens is 0.48 mu mol/L.
4. The multiplex nested PCR detection method for detecting the non-disease detection purpose of five typical pathogens of the cultured prawns by using the primer group as claimed in claim 1 is characterized by comprising the following steps:
(1) Constructing positive plasmid DNA as a control;
(2) Extracting DNA of a sample to be detected;
(3) And (3) performing an external amplification PCR reaction: constructing an external amplification PCR reaction system and an external amplification PCR reaction program, performing an external amplification PCR reaction to obtain an external amplification product, and taking positive plasmid DNA and sterile water of each pathogen as positive and negative controls respectively;
(4) Internal amplification PCR reaction: constructing an internal amplification PCR reaction system and an internal amplification PCR reaction program, and carrying out an internal amplification PCR reaction on the diluted external amplification product to obtain an internal amplification product;
(5) And (3) judging a PCR result: and detecting an internal amplification product by adopting electrophoresis, wherein if a positive strip with the same size as the positive control fragment appears, the sample carries a corresponding target pathogen.
5. The method according to claim 4, characterized in that: the external PCR reaction system in the step (3) is a 25 mu L reaction system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the external amplification upstream and downstream primers of the five pathogens is 0.32 mu mol/L.
6. The method according to claim 4, characterized in that: the procedure of the external PCR reaction described in the step (3) is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 61℃for 1min, extension at 72℃for 1min, and 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
7. The method according to claim 4, characterized in that: the internal amplification PCR reaction system in the step (4) is a 25 mu L reaction system, wherein: dNTP concentration of 1.5mmol/L, EX Taq enzyme 1.5U, SHIV, EHP, WSSV, IHHNV and VP AHPND The concentration of the inner amplification upstream and downstream primers of the five pathogens is 0.48 mu mol/L.
8. The method according to claim 4, characterized in that: the internal amplification PCR reaction procedure in the step (4) is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, and 35 cycles were performed; finally, the extension is carried out for 10min at 72 ℃.
9. The primer group as claimed in claim 1 and the kit as claimed in claims 2 and 3 are applied to the detection of five typical pathogenic non-diseases of cultured prawns simultaneously.
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| WO2018056803A1 (en) * | 2016-09-21 | 2018-03-29 | Universiti Malaya | Rapid detection of prawn viruses, method and kit thereof |
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