CN115786587A - Primer group for multiplex PCR (polymerase chain reaction) for simultaneously detecting 4 pathogens as well as detection method and kit thereof - Google Patents

Abstract

The invention provides a primer group for multiple PCR (polymerase chain reaction) for simultaneously detecting 4 pathogens, a detection method and a kit thereof, and relates to the technical field of biological detection. The invention researches and designs a nucleic acid rapid extraction method suitable for 4 common pathogens of mink living bodies and a primer group for multiplex PCR detection, wherein the primer group comprises an upstream primer and a downstream primer which are respectively used for detecting mink canine distemper virus, parvovirus, aleutian virus and pseudomonas aeruginosa. The nucleic acid extraction method provided by the invention is simple and rapid to operate and has a good extraction effect; the primer group, the detection system and the detection method for multiplex PCR detection have the characteristics of high efficiency, strong sensitivity, good specificity and good repeated stability, overcome the complex steps of single PCR, are convenient to operate, are simple and efficient, and have pioneering significance.

Description

Primer group for multiplex PCR (polymerase chain reaction) for simultaneously detecting 4 pathogens as well as detection method and kit thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a primer group for multiplex PCR (polymerase chain reaction) for simultaneously detecting 4 pathogens, a detection method and a kit thereof.

Background

Minks as one of important fur economic animals, since 20 th century 50 s introduction and propagation, over a million minks are currently planted, disease epidemic prevention and quarantine of mink populations are well done, and timely disease prevention is a key to guarantee efficient and healthy breeding of minks (wangbao et al, 2009). With the progress of medical research, the application of minks as animal models is becoming widespread. Research finds that the establishment of a emesis model by using minks is beneficial to the research of an anti-emesis mechanism and the screening of new drugs (Yan android and the like, 2020). In addition, minks are also ideal animal models for human influenza and embryo implantation related diseases (Sun et al, 2021, fenelon and Murphy, 2019). However, the mink for experiments has the problems of disordered sources, lack of a related disease inspection system and the like, and the pathogen carried by the mink must be controlled to realize the experimental zoonization of the mink. Therefore, the establishment and the improvement of an experimental mink screening system are promoted, a rapid and accurate detection method for pathogens carried by minks is established, and the method has important significance for ensuring the safety of experimenters, ensuring the accuracy of experimental data and promoting the industrialization of the experimental minks.

At present, a scale detection system for common diseases of minks is not sound. The early season of the year is the high-incidence stage of mink diseases, mainly including acute diarrheal diseases caused by canine distemper virus and enteritis parvovirus (Sun Winning et al, 2020; vermicum et al, 2020), hemorrhagic pneumonia caused by Pseudomonas aeruginosa, etc. (Ma Xiao Hui, 2019), and these diseases are usually characterized by fast onset and high mortality. In addition, the Aleutian mink virus has wide transmission ways (horizontal transmission and vertical transmission), high incidence rate, serious influence on the reproductive function of minks and serious economic loss to the mink breeding industry (Lu et al, 2021). The commercial immune colloidal gold test strip is mostly adopted for clinically detecting canine distemper and canine parvovirus diseases, and the method is simple, easy and quick, but has higher detection cost and is not suitable for large-scale detection of animal populations. The Countercurrent Immunoelectrophoresis (CIEP) is a gold standard (Dam-Tuxen and the like, 2014) of Aleutian disease virus detection which is internationally recognized and is widely applied to Aleutian disease detection of mink breeding plants in various countries, but an antigen preparation method used by the method is complex and expensive, and the mink breeding cost is increased. Pseudomonas aeruginosa needs to be separated in a laboratory, and PCR detection is carried out after nucleic acid is extracted by the kit, so that the detection cost is increased, and the time is consumed. Therefore, it is particularly important to develop a rapid, economic and convenient disease detection system for the frequent diseases of minks.

Polymerase Chain Reaction (PCR) is a method for in vitro enzymatic synthesis and amplification of specific DNA fragments, and detection of pathogens at the genomic level has proven to be the most effective detection means to date. Multiplex PCR (Mutiplex PCR) is developed on the basis of ordinary PCR, and is characterized in that specific amplification of a plurality of sites is simultaneously carried out in the same reaction system, so that not only is a test sample saved, but also time and labor are saved (Chengzheng et al, 2020). The multiplex PCR detection method can effectively improve the detection efficiency and reduce the detection cost. However, the multiplex PCR detection system does not simply superimpose a plurality of single PCR detection systems, but requires trial and error and optimization on a plurality of technical factors to obtain an optimal reaction system (Huang et al, 2018). The amplification template (sample nucleic acid) is particularly important, the acquisition of the nucleic acid is mostly dependent on a commercialized kit at present, the existing nucleic acid extraction kits on the market are mostly divided into a bacterial nucleic acid extraction kit and a viral nucleic acid (DNA/RNA) extraction kit, and for the disease samples of mixed infection of bacteria and viruses, different nucleic acid extraction kits can only be used for processing respectively, so that the workload of detection personnel is increased, and the detection cost is also increased. Therefore, a novel, simple and rapid method for rapidly extracting nucleic acid of bacteria and viruses in the same reaction system is developed, and the detection efficiency of the method for multiple PCR detection is greatly improved.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a primer group, a nucleic acid extraction method and a detection method for multiplex PCR (polymerase chain reaction) detection of mink canine distemper virus, parvovirus, aleutian disease virus and pseudomonas aeruginosa. The method can realize that one reaction system can simultaneously extract the nucleic acid in the mixed sample of the mink Canine Distemper Virus (CDV), the Aleutian disease virus (AMDV), the parvovirus (MEV) and the Pseudomonas Aeruginosa (PA), and the product can be directly used for detecting 4 pathogens through multiple PCR reaction, and the method is simple and efficient, good in specificity and high in sensitivity.

The technical scheme provided by the invention is as follows:

in one aspect, the present invention provides a primer set for multiplex PCR for simultaneous detection of 4 pathogens, which are mink canine distemper virus, parvovirus, aleutian virus and pseudomonas aeruginosa, comprising: the sequences of the upstream and downstream primers for detecting the canine distemper virus are shown as SEQ ID No.1 and SEQ ID No. 2; the sequences of the upstream and downstream primers for parvovirus detection are shown as SEQ ID No.3 and SEQ ID No. 4; the sequences of the upstream and downstream primers for detecting the Aleutian disease virus are shown as SEQ ID No.5 and SEQ ID No. 6; and upstream and downstream primers for detecting pseudomonas aeruginosa, the sequences of which are shown as SEQ ID No.7 and SEQ ID No. 8.

The invention designs 4 pairs of specific primers for multiple PCR reactions aiming at the gene conserved sequences of 4 common disease pathogens of minks. The method can simultaneously detect various pathogens of different types through one reaction, has high design requirements on primers, considers the amplification effect of a single primer pair, considers and avoids the mutual influence, cross reaction and the like between different primers/probes, and prevents the problems of false positive and the like. The primer combination is screened after a large number of primer combinations are tried, and the primer combination with the optimal effect is obtained.

In one aspect, the invention provides a kit for multiplex PCR detection of mink canine distemper virus, parvovirus, aleutian virus and Pseudomonas aeruginosa, comprising the primer set.

In one embodiment, the kit further comprises PCR detection reagents; preferably, the PCR detection reagent comprises PCR reaction buffer solution, reaction enzyme, dNTPs and RNase free H ₂ O。

In one embodiment, primers specific to the same pathogen may be packaged in a kit, or different primers may be packaged together. In one embodiment, the kit may further comprise other components, such as including but not limited to positive controls, negative controls, and the like. In one embodiment, the kit further comprises reagents for reverse transcription, or is used with a commercial reverse transcription kit. The kit was stored at-20 ℃.

In one embodiment, the invention provides a nucleic acid integrated extraction method for a mink canine distemper virus, parvovirus, aleutian disease virus and Pseudomonas aeruginosa mixed infection sample, which comprises the steps of uniformly mixing the mink canine distemper virus, parvovirus, aleutian disease virus and Pseudomonas aeruginosa with a lysis solution, and then incubating in a water bath, wherein the lysis solution comprises 90-110mmoL/L Tris-HCL,10-15mmoL/L EDTA and 0.1-1.0 vol% NP-40 and 0.1-1.0 vol% Tween 20. The present invention provides an extraction method for simultaneously extracting CDV, AMDV, MEV and PA nucleic acids in one reaction system.

In another aspect, the invention provides a multiplex PCR detection method for mink canine distemper virus, parvovirus, aleutian virus and pseudomonas aeruginosa for non-disease diagnostic purposes, the method comprising: and carrying out multiplex PCR amplification reaction on the PCR template by using the primer group or the kit.

In one embodiment, the method further comprises a step of simultaneously extracting total nucleic acids of a sample to be detected in a reaction system, and performing reverse transcription to obtain the PCR template. In one embodiment, the extraction comprises mixing a mixed sample of mink canine distemper virus, parvovirus, aleutian disease virus and Pseudomonas aeruginosa with a lysate comprising 90-110mmoL/L Tris-HCl and incubating in a water bath; 10-15mmoL/L EDTA;0.1-1.0 vol.% NP-40; 0.1-1.0% by volume of Tween.

In a toolIn embodiments of the body, the lysis solution comprises 1M Tris HCl (pH 8.0) at a final concentration of 100mM,0.5M EDTA (pH 8.0) at a final concentration of 10mM, tween 80, or Tween 20,0.5% (v/v); NP-40,0.5% (v/v); the rest is ddH ₂ O。

In a specific embodiment, proteinase K is added to the lysate for concentration (20 mg/mL), and the final concentration of proteinase K is 0.6mg/mL.

In one embodiment, the water bath incubation conditions are 55-60 ℃ water bath incubation for 25-35min, followed by 90-95 ℃ water bath incubation for more than 10min to inactivate the protease.

In a specific embodiment, the water bath incubation conditions are 55 ℃ water bath incubation for 30min followed by 95 ℃ water bath 10min inactivation of proteinase K.

In one embodiment, the lysate cleaved lysate is subjected to reverse transcription using a reverse transcription kit; preferably, a PerfectStart Unit RT Kit (cat # AUQ-01) reverse transcription Kit is used.

For multiplex PCR detection, the amplification conditions of 4 pathogens need to be considered to be consistent, so that the difficulty in designing primers is higher, and the designed primer combination is combined with specific reaction conditions after nucleic acid extraction reverse transcription and optimization, so that a better amplification curve can be amplified. Therefore, it is also important to optimize the reaction conditions of multiplex PCR.

In one embodiment, each primer is present in the amplification system at a final concentration of 0.1 to 1. Mu.M, when performing the multiplex PCR; preferably 0.5-0.8. Mu.M.

In one embodiment, the strip annealing temperature of the multiplex PCR amplification reaction is 50-62 ℃.

In one embodiment, the conditions of the multiplex PCR amplification reaction are: pre-denaturation at 94-95 deg.C for 8-10min, and denaturation at 94-95 deg.C for 10-20s; 15-30s at 50-62 deg.C, 25-30s at 72 deg.C, and 35-45 cycles.

In a specific embodiment, the optimal reaction system conditions for the multiplex PCR amplification reaction are: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 10min, storage at 4 ℃ for 35 cycles.

In one embodiment, the multiplex PCR detection method comprises the steps of (1) primer design and synthesis, (2) DNA/RNA extraction and reverse transcription of a sample to be detected, (3) multiplex PCR amplification in a reaction system, and (4) result judgment.

The invention adopts the improved cracking method to synchronously extract the nucleic acid (DNA/RNA) from the sample to be detected, so that the cracked product is simultaneously subjected to the reverse transcription process, and the cDNA obtained by the reverse transcription is used as the template of the multiple PCR reaction.

In one embodiment, the present invention performs the result determination by 2% agarose gel; mink canine distemper virus, parvovirus, aleutian virus and pseudomonas aeruginosa respectively have specific amplification bands at 917bp, 515bp, 366bp and 232 bp.

In one embodiment, the detection of clinical samples can be performed according to the multiplex PCR method established above to verify the feasibility of the method. It can also be determined whether the unknown sample to be tested is a single infection or a mixed infection.

In the present invention, four pathogens can be detected simultaneously by detecting live animal samples (oronasal secretions, blood and anal secretions).

In the present invention, the multiplex PCR detection method is not aimed at disease detection and/or treatment. Can be used in other applications, such as laboratory research analysis of pathogens.

Has the beneficial effects that:

the primer and the kit provided by the invention have the advantages of strong sensitivity and good specificity, the detection method of the invention has high efficiency, can realize one-time PCR analysis of a sample infected by mixed diseases and simultaneously and rapidly detect 4 pathogens, and the method has good repeatability and stability.

The optimized cracking method adopted by the invention can crack viruses and bacteria in a sample within 30min, release DNA/RNA, and obtain a nucleic acid product which can be directly used for reverse transcription; not only simplifies the nucleic acid extraction steps, but also greatly reduces the workload and the cost of nucleic acid extraction; the nucleic acid obtained by the cracking method has no significant difference from the kit in the later PCR detection effect.

The optimized real-time quantitative PCR system ensures that different amplified target fragments have similar amplification efficiency under the same PCR reaction condition; the detection method is simple, time-saving and labor-saving.

The method can achieve rapid detection, provide technical support for purification and research of pathogens of mink populations, lay a good foundation for purification of diseases of minks for experiments, fill up the blank of related technologies, and have pioneering significance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an agarose gel electrophoresis diagram of a method for establishing multiple PCR detection of nucleic acid extracted by a lysis method according to an embodiment of the present invention; m: marker;1: pseudomonas Aeruginosa (PA); 2: parvovirus (MEV); 3: aleutian virus (AMDV); 4: canine Distemper Virus (CDV); 5:4 kinds of pathogens single-tube multiplex PCR electrophoretograms;

FIG. 2 is a comparison graph of the effect of nucleic acid extracted by the lysis method and the kit provided by the embodiment of the invention; wherein, the nucleic acid electrophoresis chart extracted by the kit A is as follows: m: marker;1: pseudomonas Aeruginosa (PA); 2: parvovirus (MEV); 3: mink aleutian virus (AMDV); 4: canine Distemper Virus (CDV); 5:4 pathogen multiple PCR electrophorograms; b, nucleic acid electrophoresis chart extracted by a cracking method: the sequence is the same as above; c two nucleic acid extraction methods multiplex PCR electrophoresis comparison chart: m: marker;1: nucleic acid multiplex PCR electrophoresis chart extracted by the kit; 2: extracting multiple PCR electrophoretograms of nucleic acid by a cracking method;

FIG. 3 is a diagram illustrating the volume-optimized electrophoresis results of the reverse transcription template provided in the embodiment of the present invention; m: marker;1-6: the volumes of the reverse transcription templates are 1, 3, 5, 7 and 9 from left to right respectively;

FIG. 4 shows the volume-optimized electrophoresis results of the multiplex PCR template provided in the embodiment of the present invention; m: marker;1-6: the volumes of the PCR templates are respectively 0.5, 1, 2, 3, 4, 5 and 6 mu L;

FIG. 5 shows the concentration-optimized electrophoresis results of the multiple PCR primers provided in the embodiments of the present invention; m: marker;1-7: the volumes of the reverse transcription templates are respectively 1, 0.8, 0.6, 0.4, 0.2, 0.1 and 0 mu L from left to right;

FIG. 6 shows the multiple PCR annealing temperature optimized electrophoresis results provided by the embodiments of the present invention; m: marker;1-7: the annealing temperature is 50 deg.C, 53 deg.C, 56 deg.C, 59 deg.C, 62 deg.C, 65 deg.C, and 68 deg.C respectively;

FIG. 7 shows the electrophoresis results of the multiple PCR system specificity test provided in the embodiment of the present invention; m: DNA;1 to 9: sequentially PA, MEV, AMDV, CDV, CAV, CPIV, BVDV, IBDV and E.coli;

FIG. 8 shows the result of electrophoresis in the multiple PCR sensitivity experiment according to the embodiment of the present invention; m: DNA Marker DL2000; CDV, ADV, MEV and PA from top to bottom; 1 to 9: CDV concentration 1.38X 10 ¹⁰ ～1.38×10 ² copies/. Mu.L, MEV concentration 0.87X 10 ¹⁰ ～0.87×10 ² copies/. Mu.L, AMDV concentration 0.84X 10 ¹⁰ ～0.84×10 ² copies/. Mu.L, PA concentration 2.12X 10 ³ ～2.12×10 ^-6 cfu/μL；

FIG. 9 is a multiplex PCR detection electrophoretogram of 32 clinical samples, A-1: representing the electrophoretogram of the positive sample, A- (2-17): all are electrophoretic patterns of samples, B-1: represents the electrophoretogram of the positive sample, B- (2-17): all were electrophoretograms of the samples.

FIG. 10 shows the results of multiplex PCR electrophoresis with different combinations of primer pairs; m: DNA Marker;1 to 4: CDV, PA, ADV and MEV amplification products are respectively obtained from top to bottom; 5 to 6: amplification products of CDV;

FIG. 11 shows the results of multiplex PCR electrophoresis with different combinations of primer pairs; m: DNA Marker;1 to 4: CDV, ADV, MEV and PA amplification products are respectively obtained from top to bottom;

FIG. 12 shows the results of multiplex PCR electrophoresis with different combinations of primer pairs; m: DNA Marker;1 to 4: CDV, PA, ADV and MEV amplification products are respectively obtained from top to bottom;

FIG. 13 is a schematic view of: extracting multiple PCR electrophoresis results of nucleic acid by a conventional alkali cracking method; m: DNA Marker;1 to 2: CDV, PA, ADV and MEV amplification products are respectively obtained from top to bottom;

FIG. 14 is a schematic view of: extracting multiple PCR electrophoresis results of nucleic acid by other alkali cracking methods; m: DNA Marker;1 to 4: CDV, ADV, MEV and PA amplification products are respectively obtained from top to bottom;

FIG. 15: multiple PCR electrophoresis results of different reverse transcription products; m: DNA Marker;1 to 4: CDV, ADV, MEV and PA amplification products are respectively obtained from top to bottom.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Examples

1. Materials and methods

1.1 sample

Canine distemper virus and parvovirus were purchased from Bigemo petivir (Canine distemper, parvovirus); the Aleutian mink disease virus is offered by Shao Western group teachers of the institute of specialty of Chinese academy of agricultural sciences; pseudomonas Aeruginosa (PA) was offered by the snow teacher of the institute of specialty products of the Chinese academy of agricultural sciences. Clinical samples were collected from mink farms with peripheral outbreaks in Changii, hebei.

1.2 Primary reagents and instruments

TIANAmp Bacteria DNA Kit (cat # DP 302), TIANAmp Virus DNA/RNA Kit (cat # DP 315) were all purchased from Tiangen Biochemical technology Ltd; the reverse transcription Kit PerfectStart Unit RT Kit (cat # AUQ-01) is purchased from Beijing Quanji gold organisms; primerStar SH Premix (cat # R040A) was purchased from Takara; pUC-TA Quick Ligation Kit (cat # CW 2592S) was purchased from Yukang as a century; competent cell e.coli Trans-T1 was purchased from holo-golgi, ltd, beijing; the conventional reagents used in the experiments were analytically pure and purchased from Sigma. Gradient PCR instrument (ETC-811) purchased from Tosheng, beijing; the nucleic acid electrophoresis apparatus is purchased from six instruments factories of Beijing; the ultraviolet analyzer (JY 02S) was purchased from Beijing Junyi.

1.3 Experimental methods

1.3.1 primer design and Synthesis

4 pairs of specific primers were designed with reference to the CDV, MEV, AMDV, and PA gene sequences recorded in GenBank, and were synthesized by Biotechnology engineering (Shanghai) Inc., and the primer sequences are shown in Table 1-1.

TABLE 1 primer sequences and product lengths

1.3.2 lysis method for extracting DNA/RNA of virus and bacteria

(1) Lysate formula

The lysate formulations are shown in table 2.

TABLE 2 lysate formulations

Note: lysis solution formula (prepared at present use): mu.L of proteinase K is added into 1ml of the lysate for concentration (20 mg/ml), and the final concentration of the proteinase K is 0.6mg/ml.

(2) Nucleic acid DNA/RNA extraction by lysis

In the optimization stage of a laboratory, live vaccines and culture bacteria liquid are used as experimental objects to obtain nucleic acid DNA/RNA. Specifically, after canine distemper, parvovirus vaccine and aleutian virus solution are mixed with overnight-cultured pseudomonas aeruginosa bacterial solution, 10 mu L of mixed sample is added into 30 mu L of lysate (shown in table 2) to be suspended and mixed uniformly, and after incubation in a water bath kettle at 55 ℃ for 30min, water bath at 95 ℃ is carried out for 10min to inactivate proteinase K.

(3) Reverse transcription

The obtained cleavage products were subjected to reverse transcription using a reverse transcription kit, and the reverse transcription system is shown in Table 3.

Reaction conditions are as follows: mixing the liquid, and incubating at 50 deg.C for 5min; heating at 85 deg.C for 5s, and storing at-20 deg.C for subsequent detection. The cDNA obtained by this method was labeled as a cleavage group.

TABLE 3 reverse transcription reaction system (20. Mu.L)

1.3.3 establishment of multiplex PCR detection method

The PCR reaction system is shown in Table 4.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 10min, storage at 4 ℃ for 35 cycles.

The PCR product was subjected to agarose gel electrophoresis, 2% agarose gel electrophoresis, 120V,130mA,40min, and observation by gel imaging system.

TABLE 4 PCR reaction System

1.3.4 identification of multiplex PCR amplification products

Purifying and recovering PCR products containing CDV, MEV, AMDV and PA gene sequences, and connecting a pUC-TA vector; transformation of the ligation products into competent cells E.coli Trans-T1 by Heat shock transformation and plating on Amp ⁺ The LB plate of (1) is cultured for 16h at 37 ℃, a monoclonal colony is selected to be cultured overnight, plasmids are extracted according to the specification of a plasmid extraction kit and are subjected to PCR identification, the extracted positive plasmids are sent to Shanghai biological engineering technology Limited company for sequencing, and the results are compared with the sequences registered in GenBank for analysis.

1.4. Comparison of the extraction effects of the kits

Extracting the RNA of CDV, the DNA of MEV and the DNA of AMDV from the virus and bacteria liquid samples with the same volume according to the steps of the virus DNA/RNA extraction kit instruction; extracting the bacterial group DNA by a bacterial genome DNA extraction kit, and marking the obtained DNA/RNA as a kit group. And finally, performing multiple PCR amplification on the nucleic acid products of the lysis group and the nucleic acid products of the kit extraction group under the same conditions respectively, and comparing the effects of the two groups of nucleic acid products by 2% agarose gel electrophoresis.

1.5. Optimization of multiplex PCR reaction conditions

(1) Concentration of the reverse transcription template: and carrying out reverse transcription on the nucleic acid product obtained by the cracking method, setting the adding volume of the template to be 1, 3, 5, 7, 9 and 11 mu L gradient, carrying out multiple PCR amplification on the reverse transcription product, and observing the result through 2% agarose gel electrophoresis to determine the optimal adding amount of the reverse transcription template.

(2) Multiplex PCR template concentration: setting the volumes of the multiple PCR templates to be 0.5, 1, 2, 3, 4 and 5 mu L respectively, observing the result after 2 percent agarose gel electrophoresis, and determining the optimal template addition amount of the multiple PCR reaction.

(3) Annealing temperature: setting multiple PCR annealing temperature gradient 50 deg.c, 53 deg.c, 56 deg.c, 59 deg.c, 62 deg.c and 65 deg.c, and screening the optimal annealing temperature through agarose gel electrophoresis observation of the reaction product.

(4) Primer concentration: premixing 4 pairs of primers to 10 mu M, respectively adding 1, 0.8, 0.6, 0.4, 0.2, 0.1 and 0 mu L to perform multiplex PCR amplification, and screening out the optimal primer concentration and optimizing the multiplex PCR reaction conditions by observing the result through agarose gel electrophoresis.

1.6. Experiment of specificity

The nucleic acid extraction method 1.3.2 is used for extracting nucleic acids such as canine parainfluenza virus (CPIV), canine Adenovirus (CAV), bovine Viral Diarrhea Virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), escherichia coli (E.coli) and the like, a multiplex PCR system is used for amplification, a positive control is arranged at the same time, and 2% agarose gel electrophoresis is used for detecting a specific band.

1.7. Sensitivity test

The amplified products of CDV, MEV and AMDV (fragment size is 917bp, 515bp and 366bp respectively) are connected with pUC-TA vector to construct positive plasmid, and the concentration is measured by ultraviolet spectrophotometer and converted into plasmid copy number. Meanwhile, after 10 times of pseudomonas aeruginosa cultured by an LB liquid culture medium is diluted in a gradient manner, the colony count of an LB solid plate is carried out. And (3) extracting genome DNA from the counted bacterial liquid by a lysis method, fully and uniformly mixing the extracted DNA with the three positive plasmids according to a ratio of 1.

1.8. Clinical sample testing

Extracting virus and bacteria DNA/RNA from the disease material sample. Firstly, 11 parts of ophthalmic and nasal secretion and anal secretion of suspected diseased minks clinically delivered to Hebei Changli are collected, a small amount of sterile water is added, the mixture is shaken and then kept stand for 5min, supernatant liquid is absorbed, the mixture is centrifuged at 12000r/min for 3min, supernatant is discarded, 30 mu L of lysate (detailed formula shown in table 2) is added into precipitates for suspension and uniform mixing, after incubation at 55 ℃ for 30min, protease K is inactivated in water bath at 95 ℃ for 10min, and the treated product is subjected to reverse transcription and is detected by the established multiplex PCR method.

2. Analysis of results

2.1 establishment of multiplex PCR detection method and identification of amplified products

Extracting canine distemper, parvovirus vaccine, mink Aleutian disease virus culture solution and pseudomonas aeruginosa bacterial solution nucleic acid by adopting a lysis method, performing reverse transcription, and respectively amplifying CDV, AMDV, MEV and PA specific sequences consistent with expected sizes by using established multiplex PCR, wherein the sizes of amplified target fragments are 917bp, 515bp, 366bp and 232bp (figure 1). The sequencing result analysis after product recovery has 100% homology with GenBank accession sequence. Experimental results show that the nucleic acid extraction method and the multiple PCR pathogen detection method established in the research are feasible and have the advantages of rapidness, specificity, simplicity and the like.

2.2 comparison with the kit

The CDV, MEV, AMDV virus liquid and PA cultured overnight are respectively extracted by different methods for multiple PCR verification. The results show that the nucleic acid extracted from the lysate and the nucleic acid extracted from the kit have no significant difference in amplification effect by multiplex PCR (polymerase chain reaction) (figure 2). Therefore, the nucleic acid obtained by processing the sample by the lysis method can meet the requirement of the concentration of the nucleic acid in the sample by the multiplex PCR amplification.

2.3 multiplex PCR reaction Condition optimization

The reverse transcription template volume optimization experiment shows that when the template volume is between 5 and 11 mu L, a specific band can be amplified (figure 3); the multiplex PCR template volume optimization experiment shows that specific bands can be amplified when the template volume is between 0.5 and 5 mu L (figure 4); optimization experiments such as annealing temperature and primer concentration show that when the annealing temperature is 50-62 ℃ (figure 5) and the primer concentration is 1-0.2mmol (figure 4), specific bands can be amplified by multiplex PCR.

2.4 specificity assay

DNA/RNA is extracted from cell culture samples of canine distemper virus, parvovirus, aleutian virus, pseudomonas aeruginosa, canine parainfluenza virus and canine adenovirus which are common to minks by applying the method established by the invention, multiple PCR amplification results show that bands with expected size specificity are respectively amplified by the canine distemper virus, the parvovirus, the Aleutian virus and the pseudomonas aeruginosa, and the Canine Adenovirus (CAV), the canine parainfluenza virus (CPIV), the Bovine Viral Diarrhea Virus (BVDV), the bovine infectious rhinotracheitis virus (IBRV) and escherichia coli (E.coli) are negative (figure 7). Experimental results show that the multiple PCR detection method established by the invention has good specificity.

2.5 sensitivity test

The concentration was determined by UV spectrophotometer and converted to plasmid copy number to give a CDV of 1.38X 10 per microliter ¹⁰ copies, MEV 0.87X 10 per microliter ¹⁰ copies, ADV 0.84X 10 per microliter ¹⁰ copies; the colony counting result shows that the PA bacterial liquid is 2.12 multiplied by 10 per microliter ³ CFU, multiplex PCR validation of mixed template 10-fold gradient dilutions.

The results showed that the lowest nucleic acid detection amount of CDV was 1.38X 10 ² copies; the minimum nucleic acid detection amount of MEV was 0.87X 10 ³ copies; the minimum nucleic acid detection amount of AMDV is 0.84X 10 ⁴ copies; PA 2.12X 10 per microliter ^-1 CFU (fig. 8).

2.6 clinical sample test results

Clinical onset mink samples were collected, nucleic acid was extracted according to the method established in this study, and multiplex PCR detection was performed, with detection results showing that CDV infection rate was 44% (14/32), AMDV infection rate was 6% (2/32), MEV infection rate was 100% (32/32), and no target band was amplified by PA (fig. 9).

Comparative example 1

The invention relates to the detection of 4 different pathogens, if the most suitable primer combination for detecting multiple pathogens is to be screened at the same time, because the primer combination is matched with different reaction systems and reaction programs to realize the detection effect of multiple PCR, the design and screening work of the primer is time-consuming and labor-consuming. In the screening process, a plurality of different primer combinations are tried, and the method is not suitable in many cases and has an unsatisfactory effect. The following are some illustrative examples.

TABLE 5 control primer sequences

The control primer sequences are used for carrying out multiple PCR detection, and the results show that: the amplification products of PA primer pair No.9 and No.10 are 711bp in size, and are subjected to multiplex PCR with CDV, AMDV and MEV primers, the bands of PA, AMDV and MEV are clear and bright with the continuous increase of the template volume, but the CDV bands are deleted, and the clear target bands can be seen when CDV primer pair No.1 and No.2 are subjected to PCR amplification by using CDV primer pair alone (FIG. 10).

The amplification products of PA primer pair No.11 and No.12 are 263bp in size, and are subjected to multiplex PCR with CDV, AMDV and MEV primers, the target bands of CDV (917 bp), AMDV (515 bp), MEV (366 bp) and PA (263 bp) are weaker along with the continuous reduction of the template volume, and a hybrid band exists in the size of 100bp (FIG. 11).

The amplification product size of PA primer pair No.13 and No.14 is 747bp, and multiple PCR is carried out with CDV, AMDV and MEV primers, the bands of PA, AMDV and MEV are clear and bright with the increasing of the template volume, but the band of CDV target disappears, and the sizes of CDV and PA bands in the primer combination are close, so that the band is not easy to distinguish (FIG. 12).

Comparative example 2

In the present invention, CDV, AMDV, MEV and PA nucleic acids (DNA/RNA) are simultaneously extracted in one reaction system by a lysis method, and the obtained product is subjected to reverse transcription for PCR detection. The lysis solution used in the invention is a lysis solution combination for simultaneously extracting CDV, AMDV, MEV and PA nucleic acid for the first time. In the process of research and development, various types of lysates are tried and searched, and the effect is not ideal. The following are illustrative examples.

When the extraction of CDV, AMDV, MEV and PA nucleic acids was carried out using a conventional lysis solution (composition consisting of 1% SDS,200mM NaCl,5mM EDTA, 2mM Tris-HCl (pH 8.0)), the specific experimental procedures were as follows:

centrifuging the PA bacterial liquid cultured overnight at 10000rpm/min for 1min, and removing the supernatant; adding CDV, AMDV, and MEV virus solution, mixing, adding 5mg/ml lysozyme and 20 μ l proteinase K, standing at room temperature for 1min; adding an isovolumetric lysate, incubating at 56 ℃ for 20min, adding an isovolumetric phenol-chloroform-isopropanol (25; adding 2 times volume of anhydrous ethanol, precipitating at-20 deg.C for 10min, centrifuging at 10000rpm/min at 4 deg.C for 15min, and discarding the supernatant; adding 0.5ml 70% ethanol, centrifuging at 4 deg.C for 12min, and removing supernatant; after centrifugation to remove ethanol and drying at room temperature, 50. Mu.l of RNase-free Water was added. And carrying out multiple PCR detection on the nucleic acid product after reverse transcription.

The results are shown in FIG. 13: after the nucleic acid obtained by the cracking method is subjected to multiple PCR detection, the multiple effect is poor, a PA target strip is deleted, and a CDV strip is weak.

In addition, the present invention also contemplates other lysis methods: the components comprise (50mM NaOH,1M Tris-HCl pH 8.0) by adding 200. Mu.l 50mM sodium hydroxide into CDV, AMDV, MEV, PA mixture, splitting in 95 deg.C water bath for 30min, shaking thoroughly, adding 20. Mu.l 1M hydrochloric acid tris (hydroxymethyl) aminomethane (pH 8.0) into each tube, shaking for mixing, and centrifuging at 13000rpm/min for 5min. And (3) carrying out reverse transcription on the nucleic acid obtained in the step, carrying out multiple PCR amplification reaction, and carrying out agarose gel electrophoresis to identify the nucleic acid extraction effect.

The results show that: the nucleic acid extracted by the lysis method only amplifies AMDV and PA bands, and CDV and MEV bands are not seen, so that the nucleic acid extraction effect is poor (figure 14).

Comparative example 3

Experiments show that compared with other reverse transcription kits or reverse transcription products, after the lysate is used for obtaining the lysate, when the lysate and a commercial reverse transcription Kit PerfectStart Unit RT Kit (cargo number: AUQ-01) are purchased from Beijing all-around gold creatures to carry out reverse transcription and then carry out multiple PCR reactions, the effect is better (shown in figure 15).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims (10)

1. A primer set for multiplex PCR for simultaneous detection of 4 pathogens, wherein the pathogens are mink canine distemper virus, parvovirus, aleutian virus and pseudomonas aeruginosa, the primer set comprising:

the sequences of the upstream primer and the downstream primer for detecting the canine distemper virus are shown as SEQ ID No.1 and SEQ ID No. 2;

the sequences of the upstream and downstream primers for parvovirus detection are shown as SEQ ID No.3 and SEQ ID No. 4;

the sequences of the upstream and downstream primers for detecting the Aleutian disease virus are shown as SEQ ID No.5 and SEQ ID No. 6; and

the sequences of the upstream primer and the downstream primer for detecting pseudomonas aeruginosa are shown as SEQ ID No.7 and SEQ ID No. 8.

2. A kit for multiplex PCR detection of mink canine distemper virus, parvovirus, aleutian virus and Pseudomonas aeruginosa, comprising the primer set of claim 1.

3. The kit of claim 2, wherein preferably the kit further comprises PCR detection reagents;

more preferably, the PCR detection reagent comprises PCR reaction buffer solution, reaction enzyme, dNTPs and RNase free H ₂ O。

4. The multiplex PCR detection method of mink canine distemper virus, parvovirus, aleutian disease virus and pseudomonas aeruginosa for non-disease diagnosis purposes is characterized by comprising the following steps of: performing a multiplex PCR amplification reaction on a PCR template using the primer set of claim 1 or the kit of claim 2 or 3.

5. The method of claim 4, further comprising simultaneously extracting total nucleic acids from the sample to be tested in a reaction system, and performing reverse transcription to obtain the PCR template.

6. The method of claim 4, wherein the extracting comprises incubating a mixed sample of mink canine distemper virus, parvovirus, aleutian virus and Pseudomonas aeruginosa with a lysis solution comprising 90-110mmoL/L Tris-HCL,10-15mmoL/L EDTA,0.1-1.0 vol% NP-40,0.1-1.0 vol% Tween 20 in a water bath.

7. The method of claim 6, wherein the water bath incubation is performed under conditions of 55-60 ℃ for 25-35min followed by 95-100 ℃ for more than 10min inactivation of the protease.

8. The method of claim 4, wherein each primer is present in the amplification system at a final concentration of 0.1 to 1 μ M during the multiplex PCR; preferably 0.5-0.8. Mu.M.

9. The method of claim 4, wherein the strip annealing temperature of the multiplex PCR amplification reaction is 50-62 ℃.

10. The method of claim 8 or 9, wherein the conditions of the multiplex PCR amplification reaction are: pre-denaturation at 94-95 deg.C for 8-10min, and denaturation at 94-95 deg.C for 10-20s; 15-30s at 50-62 deg.C, 25-30s at 72 deg.C, and 35-50 cycles.

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