CN114703327B - Composition for detecting infectious laryngotracheitis virus of chickens and its application - Google Patents
Composition for detecting infectious laryngotracheitis virus of chickens and its application Download PDFInfo
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Abstract
本发明检测鸡传染性喉气管炎病毒的组合物及其应用，属于微生物的测定或检验方法其所用的组合物技术领域。本发明公开了试剂或试剂盒在检测或辅助检测鸡传染性喉气管炎病毒中的应用，所述试剂或试剂盒包括鸡传染性喉气管炎病毒引物探针，所述鸡传染性喉气管炎病毒引物探针由特异扩增含有核苷酸序列是SEQ ID No.1的DNA片段在内的鸡传染性喉气管炎病毒基因组DNA片段的引物和与核苷酸序列是SEQ ID No.1的DNA片段特异结合的探针组成。通过比较不同引物浓度、探针浓度、最佳退火温度、灵敏度以及检测特异性等方面进行研究，建立了微滴式数字PCR检测ILTV的绝对定量方法，为定量检测ILTV提供了技术支撑。
The invention discloses a composition for detecting infectious laryngotracheitis virus of chickens and its application, belonging to the technical field of microbial determination or inspection methods and compositions used therein. The invention discloses the application of a reagent or a kit in detecting or assisting in detecting infectious laryngotracheitis virus of chickens, wherein the reagent or the kit comprises an infectious laryngotracheitis virus primer probe of chickens, and the infectious laryngotracheitis virus primer probe of chickens is composed of a primer for specifically amplifying a DNA fragment of the infectious laryngotracheitis virus genome of chickens including a DNA fragment having a nucleotide sequence of SEQ ID No. 1 and a probe for specifically binding to the DNA fragment having a nucleotide sequence of SEQ ID No. 1. By comparing different primer concentrations, probe concentrations, optimal annealing temperatures, sensitivity, and detection specificity, an absolute quantitative method for detecting ILTV by droplet digital PCR is established, providing technical support for quantitative detection of ILTV.
Description
Technical Field
The invention belongs to the technical field of compositions used in a microorganism determination or detection method, and particularly relates to a composition for detecting infectious laryngotracheitis virus of chickens and application thereof.
Background
Infectious laryngotracheitis virus (infectious laryngotracheitis virus, ILTV) belongs to the genus infectious laryngotracheitis virus of the subfamily herpesviridae, mainly causing upper respiratory disease in chickens, which is mainly characterized by dyspnea, cough and expectoration of blood-containing exudates. The chicken with the disease can be seen as swelling and bleeding of throat and mucous membrane of the air duct, and erosion is formed. The virus is easy to infect chickens, once the chickens are infected by the virus, the virus is transmitted rapidly, the chickens are infected in a short time to more than 90 percent, the death rate can reach 20 percent or more, and great economic loss is caused.
In recent years, along with the common use of ILTV vaccines in chicken farms, the symptoms of the chicken flocks after ILTV infection are changed, so that the symptoms and lesions of the chicken flocks are easily confused with other respiratory diseases such as avian influenza, newcastle disease, infectious bronchitis and the like in a mild case, the identification is difficult to be carried out by the clinical symptoms alone, further laboratory methods are needed, but the intelligent qualitative detection of the ILTV identification method at present cannot be realized.
Since the advent of Polymerase Chain Reaction (PCR) detection in 1985, it has been widely used in various fields such as biology and medicine. With rapid development and technological progress of biotechnology, PCR technology has been developed to fluorescent quantitative PCR (qPCR) technology, and nowadays to microdroplet digital PCR (ddPCR) technology. The ddPCR technology divides a sample into hundreds or even millions of independent reaction units of water-in-oil type, and then carries out parallel amplification on DNA template molecules containing or not containing 1 or more copies in all independent reaction units, reads negative or positive fluorescent signals of each reaction unit after amplification is finished, adopts a terminal quantitative method to carry out analysis, and calculates template copy number of an original sample according to a Poisson distribution statistical method so as to achieve the aim of absolute quantification.
Microdroplet digital PCR (droplet DIGITAL PCR, DDPCR) is a new generation of polymerase chain reaction (polymerase chain reaction, PCR) technology that has emerged in recent years. The method comprises the steps of dividing a reaction solution into tens of thousands of nano-scale droplets by utilizing a water-in-oil technology, detecting each droplet one by utilizing a droplet analyzer after PCR amplification is finished, and analyzing the number and proportion of positive droplets according to a Poisson distribution principle in statistics to obtain the initial copy number or concentration of target molecules, thereby realizing absolute quantification of nucleic acid molecules with the low content of single copy. Compared with the traditional real-time fluorescence quantitative PCR (quantitative real-time PCR, qPCR) method, the method does not depend on a standard curve, so that the quantitative result is more accurate, and the influence of a reaction inhibitor on the system is greatly reduced.
Disclosure of Invention
The invention aims to solve the technical problem of how to realize the detection of the clinical sample of the chicken infectious laryngotracheitis virus.
To solve the above technical problem, in a first aspect, the present invention provides an application of the composition in any one of the following:
p1) detecting or assisting in detecting infectious laryngotracheitis virus of chicken;
P2) preparing a product for detecting or assisting in detecting infectious laryngotracheitis virus of chicken;
p3) detecting or assisting in detecting whether the sample to be detected is infected with chicken infectious laryngotracheitis virus;
p4) preparing a product for detecting or assisting in detecting whether a sample to be detected is infected with chicken infectious laryngotracheitis virus;
p5) detecting or assisting in detecting whether the pathogen to be detected is infectious laryngotracheitis virus;
P6) preparing a product for detecting or assisting in detecting whether the pathogen to be detected is infectious laryngotracheitis virus of chicken;
The composition comprises a chicken infectious laryngotracheitis virus primer probe,
The infectious laryngotracheitis virus primer Probe comprises a primer ILTV-F, a primer ILTV-R and a Probe ILTV-Probe,
The primer ILTV-F is single-stranded DNA with a nucleotide sequence of SEQ ID No. 2;
The primer ILTV-R is single-stranded DNA with a nucleotide sequence of SEQ ID No. 3;
The nucleotide sequence of the Probe ILTV-Probe is SEQ ID No.4.
Further, in the above application, the ILTV-F, ILTV-R, ILTV-Probe has a mass ratio of 2:2:1.
Further, in the above application, the digital PCR reagent or kit further comprises a positive standard plasmid.
Further, in the above application, the positive standard quality pellet comprises a DNA molecule having a nucleotide sequence shown in SEQ ID No. 1.
In order to solve the technical problem, in a second aspect, the invention provides a composition, which comprises the infectious laryngotracheitis virus primer probe.
Further, in the above composition, the ratio of the amounts of the ILTV-F, ILTV-R, ILTV-Probe is 2:2:1.
Further, the composition also comprises a positive standard plasmid.
Further, in the above composition, the positive standard quality pellet comprises a DNA molecule having a nucleotide sequence shown in SEQ ID No. 1.
The primer probes in the above composition may be packaged individually or in combination.
The positive standard quality granules in the above composition are packaged individually.
In order to solve the technical problems, in a third aspect, the invention provides a method for detecting or assisting in detecting infectious laryngotracheitis virus of chicken, which comprises performing digital PCR on a sample to be detected by using the composition, and determining or assisting in determining whether the sample to be detected is infectious laryngotracheitis virus of chicken or contains infectious laryngotracheitis virus of chicken or is infected with infectious laryngotracheitis virus of chicken according to the digital PCR product
Further, in the above method, the primer annealing condition used in the digital PCR is 56℃for 1 minute.
Taking a 20 mu L reaction system as an example, the optimized reaction system and the reaction procedure obtained by the invention are as follows:
The reaction system: 2X Supermix for probe (No dUTP) 10. Mu.L, 20. Mu. Mol/. Mu.L of each of the upstream and downstream primers, 1. Mu.L of 10. Mu. Mol/. Mu.L of probe, 2. Mu.L of DNA/RNA template, and 20. Mu.L of the reaction mixture were complemented with ddH 2 O.
The reaction procedure: 95 ℃ for 10min; setting the annealing temperature at 56 ℃ for 1min at 94 ℃ for 30s, and 40 cycles; 98 ℃ for 10min; ending at 4 ℃.
The product may be a reagent or a kit.
In the invention, the ILTV-Pro is a probe for specifically recognizing chicken infectious laryngotracheitis virus, the 5 'end of the ILTV-Pro is connected with a fluorescent group, and the 3' end of the ILTV-Pro is connected with a quenching group.
The fluorescent group may be selected from, but not limited to, common fluorescent groups such as FAM (5/6-carboxyfluorescein), VIC (green fluorescent protein), TET (tetra-chloro-6-carboxyfluorescein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), cy3, TAMRA (6-carboxytetramethyl rhodamine), ROX (carboxy-X-rhodamine), texas Red, LC RED640, cy5 (cyanine dye), LC RED705, FITC (fluorescein isothiocyanate), etc., and the principle of selecting the fluorescent group of the probe in the primer composition for double PCR detection is that the two fluorescent groups are different in color development.
The quenching group can be selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and Dabcy 1.
The above-described applications or methods are non-disease diagnostic applications or methods. The above applications or methods are not directed to obtaining disease diagnosis results or health status of a living human or animal body. The sample to be tested may be a sample from a non-living human or animal body, such as an environmental sample (e.g. air), a food (e.g. frozen food or fresh food).
According to the research, 1 pair of primers and a probe are designed and synthesized according to the conserved fragment sequence of the ILTV TK gene, FAM fluorescent groups are marked at the 5 'end of the probe, and BHQ1 quenching groups are marked at the 3' end of the probe. By comparing the aspects of different primer concentrations, probe concentrations, optimal annealing temperature, sensitivity, detection specificity and the like, an absolute quantitative method for detecting the ILTV by using the microdroplet digital PCR is established, and a technical support is provided for quantitative detection of the ILTV.
Cases of chicken onset caused by chicken ILTV are common, and the main clinical features are dyspnea, cough and expectoration of blood-containing exudates. At present, in the process of preventing and controlling chicken ILT, an ILTV vaccine plays a key role. However, in the case of general immunization, the chicken flock infected by ILTV still appears, and is difficult to distinguish from respiratory tract symptoms caused by IBV and AIV in clinical symptoms, and needs to be distinguished by a laboratory method, but under the condition of low viral load, the conventional method is also difficult to diagnose, and a detection method for low copy is needed to be found. The ddPCR method meets the requirements of low copy and absolute quantitative detection, so that the establishment of the ddPCR method for detecting ILTV has practical significance.
The beneficial technical effects obtained by the invention are as follows:
1. The experiment establishes a ddPCR method for absolute quantitative detection of ILTV, optimizes the concentration of a primer and a probe and the annealing temperature in the method, and discovers that the reaction has the lowest negative fluorescence amplitude (gray) and high positive droplet signal (blue) and little dispersion between positive droplets and negative droplets under the conditions that the final concentration of the primer is 1 mu mol/mu L and the concentration of the probe is 0.5 mu mol/mu L. The annealing temperature was 56 ℃, the difference in fluorescence amplitude between positive droplet signal (blue) and negative droplet signal (grey) was greatest, and the number of positive droplets obtained was greatest.
2. The ddPCR method established in the test is compared with the qPCR detection method in terms of sensitivity detection. When the ILTV plasmid DNA of the same dilution is quantitatively detected, quantitative values of ddPCR and qPCR are in linear positive correlation, and the ddPCR method is lower by 1 titer than the copy number detected by the qPCR method and is more sensitive.
3. In the method, when the concentration of the positive template is too high and the positive product amplified by ddPCR exceeds 10000 copies/. Mu.L, the system can not read the copy number, and in this case, in order to obtain the positive copy number of amplified positive more accurately, the detected copy number can be successfully obtained after the template concentration is diluted by a certain concentration. The method is more suitable for detecting clinical samples due to the low content of the target template in the samples.
Drawings
FIG. 1 is a graph showing the results of primer concentration optimization at ILTV DDPCR.
FIG. 2 is a graph of ILTV DDPCR probe concentration optimization results.
FIG. 3 is a graph of ILTV DDPCR annealing temperature optimization results.
FIG. 4 is a graph showing the result of ILTV DDPCR-specific detection.
FIG. 5 is a graph showing the result of ILTV DDPCR sensitivity detection.
FIG. 6 is a graph of ILTV qPCR sensitivity detection results.
Fig. 7 is a ILTV DDPCR standard graph.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The establishment of triple RT-PCR detection methods for chicken infectious laryngotracheitis virus (ILTV Beijing strain) is preserved in the laboratory, in literature Xie Zhiqin, etc., and is published in Chinese veterinary journal, 2017, 53 (2): 6-9. The public can obtain the biological material from the applicant according to the relevant regulations of national biosafety, and the biological material is only used for the relevant experiments of the repeated invention and can not be used for other purposes.
The infectious laryngotracheitis virus (Guangdong Yongshun vaccine strain K317, ILTV K317) was stored in the laboratory, and in the literature "Xie Zhiqin et al, the establishment of the infectious laryngotracheitis virus LAMP detection method, the livestock and veterinarian, 2012,44 (11): 52-55 et al, the K317" Xie Zhiqin et al, the establishment of the infectious laryngotracheitis virus LAMP detection method, the livestock and veterinarian, 2012,44 (11): 52-55 et al, the public obtained the biological material from the applicant according to the national biosafety regulations, the biological material was used only for the relevant experiments of the repeated invention, and the biological material could not be used for other purposes.
The construction of triple RT-PCR detection methods for chicken infectious bronchitis Virus (IBV Mass 41) is preserved in the laboratory and described in the literature Xie Zhiqin, etc., newcastle disease Virus, H9 subtype avian influenza Virus and avian pneumovirus, chinese veterinary journal, 2017, 53 (2): 6-9., which is available to the public from the applicant in accordance with the relevant regulations of national biosafety, and which is used only for the relevant experiments of the repeated invention and not as other uses.
Avian reovirus (Reo S1133) was saved by the present laboratory and was used only for the relevant experiments of the repeated invention, but not as other uses, as disclosed in the journal of chinese veterinarian, 2017, 53 (2): 6-9 ", in the establishment of triple RT-PCR detection methods for newcastle disease virus, subtype H9 avian influenza virus and avian pneumovirus, et al, literature" Xie Zhiqin.
The construction of triple RT-PCR detection methods for newcastle disease virus (NDV Lasota) is preserved in the laboratory and is disclosed in the literature Xie Zhiqin, etc., and the methods for triple RT-PCR detection of newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus, chinese veterinary journal, 2017, 53 (2): 6-9., the public can obtain the biological material from the applicant according to the relevant regulations of national biosafety, and the biological material is only used for the relevant experiments of repeated invention and can not be used for other purposes.
The avian influenza H9N2 066C strain (AIV H9N2 066C) was kept by this room, and was used only for the repetition of the experiments related to the present invention, but not for other uses, as disclosed in the journal of Chinese veterinarian, 2017, 53 (2): 6-9 ", in the establishment of triple RT-PCR detection methods for newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus by the literature" Xie Zhiqin et al.
Avian encephalomyelitis virus (AEV Van) was saved by this laboratory and established in literature "Xie Zhiqin et al, triple RT-PCR detection methods for newcastle disease virus, subtype H9 avian influenza virus and avian pneumovirus, journal of veterinarian, 2017, 53 (2): 6-9", which is publicly available from the applicant in accordance with the relevant regulations of national biosafety, and which is used only for repeated experiments related to the invention and not as other uses.
The avian metapneumovirus (APV/MN-10) was preserved by this test and was established in literature "Xie Zhiqin et al, triple RT-PCR detection methods for newcastle disease virus, subtype H9 avian influenza virus and avian pneumovirus, journal of veterinarian, 2017, 53 (2): 6-9", which is published by the public in terms of national biosafety, which is available from the applicant for use only in the relevant experiments of the repeated invention, and which is not available for other uses.
DdPCR Supermix for Probe available from Bio-rad, inc., U.S. (No dUTP (catalyst#: 1863024);
ddPCR droplet generation oil for Probes, droplet Reader Oil are available from Bio-rad, inc. of America;
EasyPure Virual DNA/RNA Kit (catalog number ER 201-01) was purchased from Beijing full gold biotechnology Co., ltd;
PCR kit, 100bp DNA Marker, etc. were purchased from Takara Bio-engineering (Dalian) Inc.
Example 1 construction of positive Standard plasmid
1.1, Primer relates to Synthesis
DdPCR primers and probes were designed using on-line https:// bioinfo.ut.ee/primer 3-0.4.0/software, the designed primer and probe sequences were aligned on-line using BLAST, FAM fluorophores were labeled at the 5 'end of the probe primers, and BHQ1 quenching groups were labeled at the 3' end. Primers were synthesized by the biological engineering (Shanghai) Co., ltd, and the primer and probe oligonucleotide sequences are shown in Table 1.
TABLE 1 primer and probe oligonucleotide sequences
1.2 Extraction of viral nucleic acids
Referring to EasyPure Virual DNA/RNA extraction kit instructions, the ILTV Beijing strain, ILTV K317 and control strains IBV Mass 41, reo S1133, NDV Lasota, AIV H9N2 0066C, AEV Van, APV/MN-10 DNA/RNA were extracted, and the extracted DNA/RNA was stored at-80℃for use.
1.3 Preparation of Standard templates
And (3) amplifying ILTV Beijing strain DNA by using designed primers ILTV-F and ILTV-R, performing 15g/L liposaccharide gel electrophoresis on amplified PCR products, cutting off gel of the target fragment after dyeing, and recovering the target fragment by using a gel recovery kit. The recovered fragment was ligated with the PMD18-T vector and transfected into E.coli DH 5. Alpha. Plasmid DNA in E.coli after transfection was extracted and identified as ILTV positive by PCR. The obtained positive plasmid was designated pMD18-T-ILTV, and pMD18-T-ILTV contained the DNA fragment shown in SEQ ID No. 1.
PMD18-T-ILTV is a positive standard plasmid for infectious laryngotracheitis virus of chickens.
Determination of OD260/OD280 value of positive plasmid DNA the concentration of positive plasmid DNA was calculated and converted to copy number by formula, copy number= (concentration. Times. A Fu Jiade roc constant)/(average molecular weight of one base pair. Times. Total length). The results indicated that the concentration of pMD18-T-ILTV plasmid was 48.8mg/mL.
Example 2, experimental methods
2.1 Establishment and optimization of ddPCR reactions
20. Mu.L of reaction system: 2X Supermix for probe (No dUTP) 10. Mu.L, 5-40. Mu. Mol/. Mu.L of each of the upstream and downstream primers, 1. Mu.L of 2.5-40. Mu. Mol/. Mu.L of probe, 2. Mu.L of standard template, and 20. Mu.L of the template were complemented with ddH 2 O. Generating droplets: the reaction liquid 20 mu L and the droplet generation oil 70 mu L are respectively added into the second row and the third row of holes of the droplet generation card DG8, a special rubber pad is covered, and the droplets are automatically generated on the first row by a droplet generator.
Sealing film: sucking the generated microdrops into a 96-well plate, covering an aluminum film on a PX1 heat sealing instrument, and sealing at 180 ℃ for 10 s.
And (3) PCR amplification: the 96-well plate with the sealing film is put on a PCR amplification instrument for amplification, and the reaction procedure is as follows: 95 ℃ for 10min; setting the annealing temperature at 94 ℃ for 30S and 50-60 ℃ for 1min (2 ℃/S) for 40 cycles; 98 ℃ for 10min; ending at 4 ℃.
Data reading and analysis: after the reaction is completed, the 96-well plate is placed on a QX200 droplet reader to read signals and conduct data analysis.
2.2 DdPCR specificity assay
The nucleic acids of ILTV BJ, ILTV K317, AIV H9N2 0066C, IBV Mass 41, reo S1133, NDV Lasota, AEV Van and APV/MN-10 are used as templates and are respectively added into a ddPCR reaction system optimized in 3.1 for specific detection.
2.3 DdPCR sensitivity and repeatability assays
The concentration of ILTV-positive plasmid DNA was determined, then diluted in a 10-fold gradient at 10 -2-10-9. Mu.L of each dilution was used as a template and added to the optimized ddPCR reaction, and the sensitivity of the ddPCR reaction was determined. And simultaneously, the fluorescence quantitative PCR detection is carried out by using the same primer, probe concentration and reagent, and the sensitivity of the two methods is compared. Three gradient diluted templates of 10 -4-10-6 were used for 3 replicates to test the reproducibility of ddPCR.
2.4 Clinical sample detection
Extracting nucleic acid of 40 chicken disease samples collected from 1 month in 2020 to 5 months in 2021, detecting with a 2.1 optimized reaction system and fluorescence PCR, and comparing consistency of detection results.
Example 3, results
3.1, Optimization of ddPCR reaction System and reaction procedure
3.1.1, Optimization of primer and Probe concentration for ddPCR reaction
In ddPCR reactions, different primer concentrations and probe concentrations were added, resulting in high positive droplet signal and less dispersion between positive and negative droplets when the upstream and downstream primer concentrations and probe concentrations were 20. Mu. Mol/. Mu.L and 10. Mu. Mol/. Mu.L, respectively, i.e., the final concentrations were 1. Mu. Mol/. Mu.L and 0.5. Mu. Mol/. Mu.L, respectively.
(1) Optimization of primer concentration for ddPCR reaction
The probe concentration was kept at 10. Mu. Mol/. Mu.L in the above reaction system, and a gradient experiment was performed by setting 4 sets of primer concentrations, respectively as the first set: forward and reverse primers 10. Mu. Mol/. Mu.L, respectively, second set: forward and reverse primers 20. Mu. Mol/. Mu.L, respectively, third set: forward and reverse primers 30. Mu. Mol/. Mu.L, respectively, fourth set: the forward primer and the reverse primer were 40. Mu. Mol/. Mu.L, respectively. The screening was performed using an equivalent concentration of 45 copies/. Mu.L of positive standard plasmid template and reaction conditions, and each group was tested 3 times.
As a result, FIG. 1 shows that A07 represents a primer concentration of 10. Mu. Mol/. Mu. L, B07, 20. Mu. Mol/. Mu. L, C07, 30. Mu. Mol/. Mu. L, D07, 40. Mu. Mol/. Mu.L, and the left-hand graph in FIG. 1 shows the number of droplets on the abscissa and the fluorescence intensity on the ordinate; the abscissa of the right graph in fig. 1 represents different annealing temperatures, and the ordinate represents the number of positive droplets obtained.
The results show that: the average copy number of the third group was 41.9copies. Thus, the optimal primer concentrations were selected to be 20. Mu. Mol/. Mu.L, respectively.
(2) The primer concentration in the reaction system was maintained at 20. Mu. Mol/. Mu.L, and 8 sets of probe concentration gradient experiments were set as the first set: probe concentration was 1. Mu. Mol/. Mu.L, second group: probe concentration was 1.25. Mu. Mol/. Mu.L, third group: probe concentration was 2.5. Mu. Mol/. Mu.L, fourth group: probe concentration was 5. Mu. Mol/. Mu.L, fifth group: probe concentration was 10. Mu. Mol/. Mu.L, sixth group: probe concentration was 15. Mu. Mol/. Mu.L, seventh group: probe concentration was 20. Mu. Mol/. Mu.L, eighth group: the probe concentration was 30. Mu. Mol/. Mu.L. The screening was performed using an equivalent concentration of 45 copies/. Mu.L of positive standard plasmid template and reaction conditions, and each group was tested 3 times.
As a result, FIG. 2 shows that A08 represents a probe concentration of 1. Mu. Mol/. Mu. L, B08 represents a probe concentration of 1.25. Mu. Mol/. Mu. L, C08, that 2.5. Mu. Mol/. Mu. L, D08 represents a probe concentration of 5. Mu. Mol/. Mu.L, E08 represents a probe concentration of 10. Mu. Mol/. Mu.L, F08 represents a probe concentration of 15. Mu. Mol/. Mu.L, G08 represents a probe concentration of 20. Mu. Mol/. Mu.L, H08 represents a probe concentration of 30. Mu. Mol/. Mu.L, and the abscissa of the left graph in FIG. 2 represents the number of droplets and the ordinate represents the fluorescence intensity. The abscissa of the right graph in fig. 2 represents different annealing temperatures, and the ordinate represents the number of positive droplets obtained.
The results show that: the copy number average of the fifth group was the highest, 42copies. Thus, the optimum probe concentration was selected to be 10. Mu. Mol/. Mu.L.
The above results indicate that: when the concentration of the upstream and downstream primers and the concentration of the probe are 20. Mu. Mol/. Mu.L and 10. Mu. Mol/. Mu.L, respectively, that is, the final concentrations are 1. Mu. Mol/. Mu.L and 0.5. Mu. Mol/. Mu.L, respectively, the signal of the positive microdroplet is high and the dispersion between the positive microdroplet and the negative microdroplet is small.
3.1.2 Optimization of annealing temperature for ddPCR reaction
In the ddPCR reaction, when the concentration of the primer and the concentration of the probe at the upstream and downstream are 20. Mu. Mol/. Mu.L and 10. Mu. Mol/. Mu.L, respectively, and the concentration of the template is about 800 copies/. Mu.L, the amplification was performed by the ddPCR reaction at the annealing temperatures of 50, 51, 52, 54, 55, 56, 58 and 60℃in 8 different gradients, and as a result, the number of positive droplets was the highest when the annealing temperature was 56 ℃. As a result, FIG. 3 shows that in FIG. 3, A04 represents 50 ℃, B04 represents 51 ℃, C04 represents 52 ℃, D04 represents 54 ℃, E04 represents 55 ℃, F04 represents 56 ℃, G04 represents 58 ℃, and H04 represents 60 ℃. The abscissa of the left graph in fig. 3 represents the number of droplets, and the ordinate represents the fluorescence intensity; the abscissa of the right graph in fig. 3 represents different annealing temperatures, and the ordinate represents the number of positive droplets obtained.
When the annealing temperature is 55-58 ℃, the positive droplets appear blue, the negative droplets appear black, the difference in fluorescence amplitude between the positive droplet signal (blue) and the negative droplet signal (gray) is greatest, and the number of positive droplets obtained is greatest. Combining these factors, the optimal annealing temperature is 56 ℃.
The results in FIG. 3 show that the optimum annealing temperature is 55-58 ℃.
3.1.3, Optimized reaction System and reaction procedure
20. Mu.L of reaction system: 2X Supermix for probe (No dUTP) 10. Mu.L, 20. Mu. Mol/. Mu.L of each of the upstream and downstream primers, 1. Mu.L of 10. Mu. Mol/. Mu.L of probe, 2. Mu.L of DNA/RNA template, and 20. Mu.L of the reaction mixture were complemented with ddH 2 O. The reaction procedure: 95 ℃ for 10min; setting the annealing temperature at 56 ℃ for 1min (2 ℃/S) at 94 ℃ for 30S, and 40 cycles; 98 ℃ for 10min; ending at 4 ℃.
3.2, DdPCR reaction specificity test results
The established ddPCR reaction system is used for carrying out ddPCR amplification by taking the nucleic acids of ILTV BJ, ILTV K317, AIV H9N2 066C, IBV Mass 41, reo S1133, NDV Lasota, AE Van and APV/MN-10 as templates. The results are shown in FIG. 4, and A06 in FIG. 4 represents the Beijing strain of ILTV; b06 represents ILTV K317; c06 represents AIV H9N2 0066C; d06 denotes IBV Mass 41; e06 represents Reo S1133; f06 represents NDV LaSota; g06 represents AE Van; h06 represents APV/MN-10.
In fig. 4, the abscissa of the left graph represents the number of droplets, the ordinate represents the fluorescence intensity, the abscissa of the right graph represents the sample number, and the ordinate represents the number of positive droplets.
The results in fig. 4 show that: the total amplified droplets in each well have more than 1 ten thousand of generated amount, and are balanced, the droplet amplification conditions are satisfied, the samples with positive droplets are ILTV BJ and ILTV K317, and other samples have no positive droplets.
3.3, DdPCR sensitivity and repeatability determination results
The results of adding 10 -2-10-9. Mu.L of each dilution as template to the optimized ddPCR reaction are shown in FIG. 2.
FIG. 5A 03 shows ILTV DNA at a dilution of 10 -2; b03 represents ILTV DNA at a dilution of 10 -3; c03 represents ILTV DNA at a dilution of 10 -4; d03 represents ILTV DNA at a dilution of 10 -5; e03 represents ILTV DNA at a dilution of 10 -6; f03 represents ILTV DNA at a dilution of 10 -7; g03 represents ILTV DNA at a dilution of 10 -8; h03 represents ILTV DNA at a dilution of 10 -9.
The abscissa of the left graph in fig. 5 represents the number of droplets, and the ordinate represents the fluorescence intensity; the abscissa of the right graph in fig. 5 represents sample numbers, and the ordinate represents positive droplet copy numbers;
The qRT-PCR results are shown in FIG. 6, and only 10 -6 dilutions were detected, low copy 4.6 copies/. Mu.L (10 -7 dilutions) was not detected.
The results show that: the lowest limit of detection for ddPCR was 4.6 copies/. Mu.L (10 -7 dilution), whereas qPCR only detected 10 -7 dilution, low copy 4.6 copies/. Mu.L (10 -8 dilution) was not detected.
The standard curve was plotted with the logarithmic values of nucleic acid dilution and positive copy number 10, respectively, and found that the linear equation was y= -1.06x+8.18, and the linear relationship R 2 value was 0.9979, and the result was shown in fig. 7. The abscissa in fig. 7 represents the logarithmic value of the sample dilution, and the ordinate represents the logarithmic value of the positive copy number obtained.
Three groups of DNA templates (10 -4-10-6 pMD18-T-ILTV plasmids diluted in a gradient manner) with different copy number concentrations are adopted to evaluate the repeatability of the ddPCR method, the test results are shown in table 2, the variation coefficient of each dilution is less than 5%, and the method has the advantages of good repeatability, high accuracy and stable and reliable detection result.
TABLE 2 ddPCR repetition test results
3.4 Detection results of clinical samples
For detection of 40 chicken disease clinical samples, the dd PCR detection result shows that 5 samples are ILTV positive, the nucleic acid copy number is 5.5-729 copies/. Mu.L, and 35 samples are negative. The qPCR assay was positive for ILTV in 4 samples and negative for 36 samples. qPCR detected that all of 4 positive samples dd PCR were positive, and 1 sample qPCR with low positive dd PCR was not detected.
TABLE 3 detection results of clinical samples
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Sequence listing
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<120> Composition for detecting infectious laryngotracheitis virus of chicken and application thereof
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