CN116377099A - Primer, kit and method for detecting fever with hemorrhagic disease pathogen - Google Patents
Primer, kit and method for detecting fever with hemorrhagic disease pathogen Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a method for detecting a fever with hemorrhagic disease pathogen, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO.262. According to the invention, 131 pairs of specific primers are designed according to the highly conserved regions of 26 pathogens of the fever with hemorrhage related diseases, and 26 fever with hemorrhage pathogen detection methods are established, and the detection methods can cover the detection of the pathogens of the fever with hemorrhage related diseases relatively completely, and have the advantages of high accuracy, strong specificity, high sensitivity, short TAT15h rapid detection, reduced mortality and improved accurate medication.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a kit and a method for simultaneously detecting a pathogen with fever and hemorrhagic disease.
Background
Fever with bleeding is a general term for fever (more than or equal to 37.5 ℃ C., the course of disease is less than or equal to 3 weeks) accompanied by a series of diseases with the following clinical manifestations of 2 or more (skin bleeding point purpura, mucosal bleeding point, epistaxis, hemoptysis, bloody stool, anemia, thrombocytes lower than normal level and continuously reduced, other bleeding manifestations). Clinically, diseases causing fever with bleeding mainly include dengue fever (dengue fever), hemorrhagic fever with renal syndrome (hemorrhagic fever with renal syndrome, HFRS), fever with thrombocytopenia syndrome (severe fever with thrombocytopenia syndrome, SFTS), plague, leptospirosis (LS) disease, swine streptococcosis, and the like. Pathogens include viruses and bacteria 2 major classes, mainly including dengue virus (DENV), hantavirus (HTNV), new Bunyavirus (NBYV), leptospira, streptococcus suis, and the like. Such pathogens cause serious infections and diseases of the hematopoietic system, posing a great threat to human health.
The traditional etiology diagnosis is determined by the morphological, physiological and biochemical reactions and immunological characteristics of pathogens, the required time is long, the operation process is complicated, and the pathogens are difficult to be identified due to the little understanding of pathogenic microorganisms and the wide application of clinically broad-spectrum antibiotics. The product aiming at fever with bleeding in the current market is mainly an ELISA detection kit for fever with thrombocytopenia syndrome based on an immune method or a nucleic acid detection kit for thirteen fever with bleeding pathogens based on a fluorescent PCR method.
With the development of molecular biology technology, second generation sequencing technology has become an indispensable means for clinical one-stop identification of pathogens. The targeted high-throughput sequencing technology (tNGS) is a solution for detecting multiple pathogens by combining a new generation sequencing platform (NGS) through a multiple PCR technology, and is characterized by short period, high detection precision and wide coverage range. Currently, there is no primer-based nucleic acid detection product for fever with hemorrhagic disease based on tNGS.
Disclosure of Invention
Based on this, the present invention aims to provide a primer, a kit and a method for detecting a pathogen associated with fever with hemorrhage.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the present invention, there is provided a primer for detecting a causative agent of fever with bleeding, comprising:
primers for detecting yersinia pestis, the sequences of which are shown as SEQ ID NO. 1-SEQ ID NO. 12;
primers for detecting streptococcus suis, the sequences of which are shown as SEQ ID NO. 13-SEQ ID NO. 24;
a primer for detecting vibrio vulnificus, which is shown as SEQ ID NO. 25-SEQ ID NO. 36;
primers for detecting aeromonas with sequences shown as SEQ ID NO. 37-SEQ ID NO. 46;
primers with sequences shown in SEQ ID NO. 47-SEQ ID NO.56 for detecting leptospira genus (leptospira wild);
primers with sequences shown in SEQ ID No. 57-SEQ ID No.68 for detecting leptospira genus (leptospira renifolia);
primers for detecting hantavirus with sequences shown as SEQ ID NO. 69-SEQ ID NO. 74;
primers for detecting the Hancheng virus with the sequences shown as SEQ ID NO. 75-SEQ ID NO. 88;
primers for detecting California encephalitis virus, the sequences of which are shown as SEQ ID NO. 89-SEQ ID NO. 100;
primers for detecting the rift valley fever virus, the sequences of which are shown as SEQ ID NO. 101-SEQ ID NO. 110;
primers for detecting Crimedes-Congo hemorrhagic fever viruses, with the sequences shown in SEQ ID No. 111-SEQ ID No. 118;
a primer for detecting the novel bunyavirus, the sequence of which is shown as SEQ ID NO. 119-SEQ ID NO. 126;
primers for detecting yellow fever virus with the sequences shown as SEQ ID NO. 127-SEQ ID NO. 134;
primers for detecting dengue virus, the sequences of which are shown as SEQ ID NO. 135-SEQ ID NO. 140;
a primer for detecting Japanese encephalitis virus, the sequence of which is shown as SEQ ID NO. 141-SEQ ID NO. 150;
primers for detecting West Nile virus with sequences shown as SEQ ID NO. 151-SEQ ID NO. 158;
primers for detecting St.Louis encephalitis virus with sequences shown in SEQ ID NO. 159-SEQ ID NO. 166;
primers for detecting the ursavirus shown in SEQ ID NO. 167-SEQ ID NO. 174;
primers for detecting the forest encephalitis virus, the sequences of which are shown as SEQ ID NO. 175-SEQ ID NO. 180;
primers for detecting Zika virus, the sequences of which are shown as SEQ ID NO. 181-192;
primers for detecting ALKhurma hemorrhagic fever virus with the sequence shown in SEQ ID NO. 193-SEQ ID NO. 200;
primers for detecting lymphocytic choriomeningitis viruses, the sequences of which are shown as SEQ ID NO. 201-SEQ ID NO. 208;
primers for detecting the Laxavirus, the sequences of which are shown as SEQ ID NO. 209-SEQ ID NO. 218;
primers for detecting mammal arenavirus (dove virus) shown in SEQ ID NO. 219-SEQ ID NO. 226;
primers for detecting mammal arenavirus (guarana virus) as shown in SEQ ID NO. 227-SEQ ID NO. 234;
primers for detecting mammal arenavirus (Ma Qiubo virus) with the sequence shown as SEQ ID NO. 235-SEQ ID NO. 242;
primers for detecting Ebola virus, the sequences of which are shown as SEQ ID NO. 243-SEQ ID NO. 246;
the sequence is shown as SEQ ID NO. 247-SEQ ID NO. 254.
The sequence is shown as SEQ ID NO. 255-SEQ ID NO.262 and is used for detecting the primer of the Casinolin disease virus.
In a second aspect of the invention, there is provided a kit for detecting a causative agent of fever with bleeding, comprising the above primer for detecting a causative agent of fever with bleeding.
In a third aspect of the invention, there is provided a method of detecting a causative agent of fever with bleeding, comprising the steps of: and (3) carrying out multiplex PCR amplification by taking the nucleic acid of the sample to be detected as a template and the primer for detecting the fever with hemorrhagic disease pathogen as a primer.
According to the invention, 131 pairs of specific primers are designed according to the highly conserved regions of 26 pathogens of the fever with hemorrhage related diseases, and 26 fever with hemorrhage pathogen detection methods are established, and the detection methods can cover the detection of the pathogens of the fever with hemorrhage related diseases relatively completely, and have the advantages of high accuracy, strong specificity, high sensitivity, short TAT15h rapid detection, reduced mortality and improved accurate medication.
Drawings
FIG. 1 shows the results of the detection limit test of the method of the present invention in the test example of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: ALaboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
In the present invention, according to 26 pathogens of fever with bleeding related diseases (including 21 viruses-Hantaan virus, hancheng virus, california encephalitis virus, rift valley fever virus, crimea-Congo hemorrhagic fever virus, novel bunyavirus, yellow fever virus, dengue virus, japanese encephalitis virus, west Nile virus, st.Louis encephalitis virus, usu graph virus, forest encephalitis virus, zika virus, ALKhura hemorrhagic fever virus, kazakhstan forest disease virus, lymphocytic choriomeningitis virus, lassa virus, mammalian arenavirus (Guanarto virus (GTOV), vernonin virus (JUNV), ma Qiubo virus (MACV)), ebola virus, sand fly virus, and 5 bacteria, namely streptococcus suis, yersinia pestis, vibrio vulnificus, aeromonas, leptospira (leptospira reniforme, leptospira wild) are used for completely covering pathogens of diseases related to fever with bleeding, 131 pairs of specific primers are designed aiming at highly conserved regions of the pathogens, 26 nucleic acid detection methods of the fever with bleeding pathogens are established, firstly, PCR amplification is carried out in an amplification tube to enrich target pathogens (the concentration ratio of primer pool of multiplex PCR is adjusted), so that finally sequencing of each target gene region can generate sequencing data with higher uniformity, the targeted amplification flow can be adapted to DNA/RNA, DNA and RNA pathogens can be detected at one time, the cost is reduced, the complexity of detection is reduced), and sequencing joints of sample sources are distinguished through the second round of PCR connection, then sequencing is carried out on a KM MiniSeqDx-CN (three registration certificates are obtained) sequencing platform, and finally, the sequencing result is subjected to belief analysis. Because the time of the infectious disease is life, compared with the detection duration of 24 hours of the metagene sequencing technology, the rapid detection can be completed by the method as short as 15 hours of TAT, the death rate is reduced, and the accurate medication is improved.
In one embodiment of the present invention, a primer for detecting a causative agent of fever with bleeding is disclosed, comprising:
primers for detecting yersinia pestis, the sequences of which are shown as SEQ ID NO. 1-SEQ ID NO. 12;
primers for detecting streptococcus suis, the sequences of which are shown as SEQ ID NO. 13-SEQ ID NO. 24;
a primer for detecting vibrio vulnificus, which is shown as SEQ ID NO. 25-SEQ ID NO. 36;
primers for detecting aeromonas with sequences shown as SEQ ID NO. 37-SEQ ID NO. 46;
primers with sequences shown in SEQ ID NO. 47-SEQ ID NO.56 for detecting leptospira genus (leptospira wild);
primers with sequences shown in SEQ ID No. 57-SEQ ID No.68 for detecting leptospira genus (leptospira renifolia);
primers for detecting hantavirus with sequences shown as SEQ ID NO. 69-SEQ ID NO. 74;
primers for detecting the Hancheng virus with the sequences shown as SEQ ID NO. 75-SEQ ID NO. 88;
primers for detecting California encephalitis virus, the sequences of which are shown as SEQ ID NO. 89-SEQ ID NO. 100;
primers for detecting the rift valley fever virus, the sequences of which are shown as SEQ ID NO. 101-SEQ ID NO. 110;
primers for detecting Crimedes-Congo hemorrhagic fever viruses, with the sequences shown in SEQ ID No. 111-SEQ ID No. 118;
a primer for detecting the novel bunyavirus, the sequence of which is shown as SEQ ID NO. 119-SEQ ID NO. 126;
primers for detecting yellow fever virus with the sequences shown as SEQ ID NO. 127-SEQ ID NO. 134;
primers for detecting dengue virus, the sequences of which are shown as SEQ ID NO. 135-SEQ ID NO. 140;
a primer for detecting Japanese encephalitis virus, the sequence of which is shown as SEQ ID NO. 141-SEQ ID NO. 150;
primers for detecting West Nile virus with sequences shown as SEQ ID NO. 151-SEQ ID NO. 158;
primers for detecting St.Louis encephalitis virus with sequences shown in SEQ ID NO. 159-SEQ ID NO. 166;
primers for detecting the ursavirus shown in SEQ ID NO. 167-SEQ ID NO. 174;
primers for detecting the forest encephalitis virus, the sequences of which are shown as SEQ ID NO. 175-SEQ ID NO. 180;
primers for detecting Zika virus, the sequences of which are shown as SEQ ID NO. 181-192;
primers for detecting ALKhurma hemorrhagic fever virus with the sequence shown in SEQ ID NO. 193-SEQ ID NO. 200;
primers for detecting lymphocytic choriomeningitis viruses, the sequences of which are shown as SEQ ID NO. 201-SEQ ID NO. 208;
primers for detecting the Laxavirus, the sequences of which are shown as SEQ ID NO. 209-SEQ ID NO. 218;
primers for detecting dove's virus (mammal arenavirus) shown in SEQ ID NO. 219-SEQ ID NO. 226;
primers for detecting melon Ruitovirus (mammal arenavirus) with the sequence shown in SEQ ID NO. 227-SEQ ID NO. 234;
primers for detecting equine autumn wave virus (mammal arenavirus) with sequences shown as SEQ ID NO. 235-SEQ ID NO. 242;
primers for detecting Ebola virus, the sequences of which are shown as SEQ ID NO. 243-SEQ ID NO. 246;
primers for detecting sand fly virus with the sequence shown in SEQ ID NO. 247-SEQ ID NO. 254;
the sequence is shown as SEQ ID NO. 255-SEQ ID NO.262 and is used for detecting the primer of the Casinolin disease virus.
In other embodiments of the invention, the use of the primer for detecting a febrile hemorrhagic disease pathogen described above in the preparation of a kit for detecting a febrile hemorrhagic disease pathogen is disclosed.
In other embodiments of the invention, a kit for detecting a febrile concomitant hemorrhagic disease pathogen is disclosed, comprising the above-described primer for detecting a febrile concomitant hemorrhagic disease pathogen.
In some of these embodiments, the kit further comprises Multi-PCRbuffer and RT Enzyme.
In some embodiments, the working concentration of the primers for detecting Yersinia pestis, streptococcus suis, vibrio vulnificus, aeromonas dactylifera, aeromonas hydrophila is 180 nM-220 nM; the working concentration of the primer for detecting leptospira (leptospira wild and leptospira kidney) is 230 nM-280 nM; the primers for detection of hantavirus, hanchen virus, california encephalitis virus, rift valley fever virus, crimia-congo hemorrhagic fever virus, novel bunia virus, yellow fever virus, dengue virus, japanese encephalitis virus, west nile virus, st.Louis encephalitis virus, urso virus, forest encephalitis virus, zika virus, ALKhurma hemorrhagic fever virus, lymphocytic choriomeningitis virus, lassa virus, mammalian arenavirus (dove virus, guars virus, equine autumn wave virus), ebola virus, sand fly virus, cassino-karst virus were all at a working concentration of 380nM to 420nM.
In other embodiments of the present invention, there is provided a method of detecting a causative agent of fever with bleeding, comprising the steps of: and (3) carrying out multiplex PCR amplification by taking cDNA of a sample to be detected as a template and the primer for detecting the fever with hemorrhagic disease pathogen as a primer.
In some embodiments, the cDNA is obtained by reverse transcription using RNA of the sample to be tested as a template, and the reaction system of the reverse transcription is: template RNA 14. Mu.l, random hexamers 2. Mu.l, 10 XRT Mix 2. Mu.l, reverse transcriptase 2. Mu.l; the reaction procedure of reverse transcription is as follows: 25 ℃ for 5min,37 ℃ for 45min and 85 ℃ for 5s.
In some of these embodiments, the multiplex PCR amplification reaction system comprises: 5x Multi-PCRbuffer 5. Mu.l, RT Enzyme Mix 3. Mu.l, primer pool 7.5. Mu.l, cDNA9.5. Mu.l.
In some of these embodiments, the multiplex PCR amplification reaction procedure is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 60℃for 1min, extension at 72℃for 1min,28 cycles; after the cycle is completed, the extension is carried out for 1min at 72 ℃.
In some embodiments, the multiplex PCR amplification is followed by a second round of PCR amplification, the reaction system of the second round of PCR amplification comprising: 25. Mu.l of PCR mix, 52.5. Mu.l of Index N, 72.5. Mu.l of Index N, and 20. Mu.l of multiplex PCR amplification product; the reaction procedure of the second round of PCR amplification is as follows: 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 60℃for 15s, extension at 72℃for 30s,10cyc; and after the circulation is finished, the temperature is 72 ℃ and the extension is carried out for 5min.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 primers for detection of fever with hemorrhagic pathogen
The Primer3 is applied to biological software, primers are designed aiming at the conserved regions of 26 pathogens related to fever with hemorrhagic disease, so that the designed primers are ensured to be specific primers, a pair of primers can only amplify corresponding sequence fragments, and no interference, namely no homology and complementarity, exists between the primers and other primers, and a hairpin structure is not formed. The screening principle is as follows:
1. designing as many available primers as possible for each target region as alternatives;
2. according to the specificity of primer amplification, whether the primer contains simple repeated sequences and whether high-frequency SNP loci exist on the primer;
3. for the primers meeting the filtering condition of the last step, selecting a pair of primers with highest Primer3 score from each target region, wherein the primers selected from different target regions meet the principle of minimizing the interaction between the primers, namely minimizing the 3' -end of the Primer and minimizing the overall complementarity ratio of the primers.
Then, a linker sequence tcgtcggcagcgtcagatgtgtataagagacag is added to the 5 'end of each forward primer, a linker sequence gtctcgtgggctcggagatgtgtataagagacag is added to the 5' end of each reverse primer, and the amplification product is 100-200bp. Specific sequences of the specific primers are shown in Table 1.
TABLE 1 specific primers for 26 pathogens associated with fever with bleeding
Example 2 method of detecting causative agent of fever with bleeding
The method comprises the following steps:
1. extraction of nucleic acids
1. Cerebrospinal fluid pretreatment
a) Placing the sampling tube on a vortex mixer, fully vortex for 30 seconds, sucking 1.3mL of sample, and carrying out high-speed centrifugation to enrich pathogens, wherein the rotating speed is 12000rpm, and the time is 5min;
b) Removing the supernatant after centrifugation, reserving 400 mu L of sample, and blowing and uniformly mixing;
c) Taking 400 mu L of sample to a bead mill tube of an extraction kit, adding 40 mu L of SDS, screwing a cover, and putting into a wall breaking instrument to perform wall breaking treatment (4700 rpm, oscillating for 45s, stopping for 20s, and performing 2 times at intervals, and performing co-oscillating for 3 times for 135 s);
d) After the wall breaking treatment is finished, centrifuging at 12000rpm for 5min, and taking 400 mu L (manual extraction) or 250 mu L (automatic extraction) of supernatant for nucleic acid extraction;
2. nucleic acid extraction
Manual extraction was performed using "nucleic acid extraction or purification reagent" (Guangzhou Meiyi Biotechnology Co., ltd.; cat# R6672-01B); or by using "nucleic acid extraction or purification reagents" (Guangzhou Meiyi Biotechnology Co., ltd., product No. R6672B-F-24, R6672B-F-48, R6672B-F-96) on an automated extraction workstation KingFisher flex. And meanwhile, the water without the nuclease is used as pure water to control NTC1 to be extracted together with a sample so as to control the pollution of the whole test.
3. Nucleic acid concentration detection
Sample nucleic acid concentrations were determined using Qubit 3.0/4.0, operated as per Equalbit DNAHSAssay Kit instructions, and the nucleic acid concentrations were recorded. Or using a spectrophotometer to determine the concentration of the sample nucleic acid, operating according to the instructions of the spectrophotometer, and recording the concentration of the nucleic acid.
2. Enrichment of target fragments
1. Primer pool
131 pairs of primers (SEQ ID NO. 1-SEQ ID NO. 262) of Table 1 were mixed to form Primer Pool with concentrations referenced to Table 1 (SEQ ID NO. 1-SEQ ID NO.46 Primer concentration 200nM,SEQ ID NO.47-SEQ ID NO.68 Primer concentration 250nM,SEQ ID NO.69-SEQ ID NO.262 Primer concentration 400 nM). Primer Pool was used for the first round of PCR, i.e. enrichment of the nucleic acid fragment of interest, and addition of the adaptor sequences required for the second round of amplification.
2. Reverse transcription
Reverse transcription was performed using the RNA extracted in the first step as a template using a reverse transcription reagent from NEB company, and the reverse transcription reaction system and the procedure are shown in Table 2.
TABLE 2
3. First round multiplex PCR amplification
The first round multiplex PCR amplification was performed using the DNA extracted in the first step or the reverse transcribed cDNA as a template and the primer pool as a primer, and the reaction system and the procedure are shown in Table 3.
TABLE 3 Table 3
Mu.l of ddH was added to 25. Mu.l of the PCR product 2 O, mixing uniformly, adding 50 mu l AMpure XP Beads, blowing and mixing uniformly, and standing at room temperature for 5min; placing on a magnetic rack, clarifying for 5min, and discarding supernatant; adding 200 μl of newly prepared 80% ethanol, standing on a magnetic rack for 30s, discarding supernatant, and repeating for one time; sucking residual ethanol, uncovering, airing at room temperature for about 5min, and adding 23 μl ddH after the magnetic beads are dried 2 O eluting DNA; after mixing by blowing, standing for 3min at room temperature, and sucking 20 μl for secondary amplification.
4. Second round PCR amplification
The second PCR amplification (using NEB secondary amplification Index primer) was performed using the first PCR product as a template, and the reaction system and procedure are shown in Table 4.
TABLE 4 Table 4
Adding 50 mu l AMpure XP Beads into 50 mu l of PCR product, blowing and mixing uniformly, and standing at room temperature for 5min; placing on a magnetic rack, clarifying for 5min, and discarding supernatant; adding 200 μl of newly prepared 80% ethanol, standing on a magnetic rack for 30s, discarding supernatant, and repeating for one time; sucking residual ethanol, uncovering, airing at room temperature for about 5min, and adding 23 μl ddH after the magnetic beads are dried 2 O eluting DNA; after mixing by blowing, standing for 3min at room temperature, and sucking 21 μl for secondary amplification.
5. Sequencing library pooling and on-machine sequencing
The purified product after the second PCR amplification was pooling at 50ng per sample. quantitative qubit, quality inspection qualified library and Miniseq sequencing.
Test example 1 methodological verification of the detection method of the present invention
1. Accuracy of
And (5) judging the accuracy of the detection result by adopting the positive coincidence rate. By adopting the detection method of example 2, 30 cases of clinical "metagene sequencing" detection items are detected, and samples with positive detection results are determined to be detected, and consistency between comparison and known results is achieved. The positive compliance results are shown in table 5.
TABLE 5 Positive compliance rate
As shown in the results of Table 5, all samples were qualified in quality control in 30 positive samples, and the positive coincidence rate was 100% in 30 cases.
2. Specificity (specificity)
And (5) carrying out specificity judgment on the detection result by adopting a negative coincidence rate (Negative Coincidence Rate). By adopting the detection method of example 2, 17 cases of clinical "metagene sequencing" detection items are detected, and samples with negative detection results are detected, and the consistency between the detection results and known results is compared. The negative compliance results are shown in table 6.
TABLE 6 negative compliance rate
As is clear from the results in Table 6, in 17 negative samples, all samples were qualified for quality control, all the samples were detected as negative, and the negative coincidence rate was 100%.
3. Detection limit
The results are shown in FIG. 1. The results in FIG. 1 show that the concentration is 10 2 copy/ml, all pathogens can be detected, so the minimum detection limit is 10 2 copy/ml。
Test example 2 primer concentration optimization test
26 samples were selected, corresponding to 26 pathogens in the present invention, respectively, and 131 pairs of primers in the primer pool of the first round of PCR corresponded to 131 amplicon numbers. The primers were divided into 3 groups, M1 group comprising primers SEQ ID NO. 1-SEQ ID NO.46, M2 group comprising primers SEQ ID NO. 47-SEQ ID NO.68, and M3 group comprising primers SEQ ID NO. 69-SEQ ID NO.262.3 groups of primers are respectively proportioned according to the concentration of the primers. According to the detection method of example 2 of the present invention, the amplification efficiency of the primer is judged based on the detection of the amplicon.
TABLE 7
As can be seen from the results in Table 7, each amplicon was detected using scheme 2, i.e., M1 (SEQ ID NO. 1-SEQ ID NO. 46) at 200nM, M2 (SEQ ID NO. 47-SEQ ID NO. 68) at 250nM, and M3 (SEQ ID NO. 69-SEQ ID NO. 262) at 400 nM.
Experimental example 3 optimization test of reaction procedure
Using the method of example 2 of the present invention, 1 sample containing 4 pathogens (Streptococcus suis, vibrio vulnificus, aeromonas, leptospira renis) was tested according to the PCR cycle numbers of 25cyc, 26cyc, 28cyc, 30cyc, 32cyc, respectively, in multiplex PCR amplification (sample 1-4). Library concentrations and detected pathogens are shown in table 8.
TABLE 8
As can be seen from the results in Table 8, the library concentrations of 25cyc and 26cyc were low, and some pathogens could not be detected. 28cyc-32cyc each pathogen was detected while ensuring sufficient library concentration. In order to shorten the time as much as possible, the optimum cycle number was set to 28cyc.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A primer for detecting a causative agent of fever with bleeding, comprising:
primers for detecting yersinia pestis, the sequences of which are shown as SEQ ID NO. 1-SEQ ID NO. 12;
primers for detecting streptococcus suis, the sequences of which are shown as SEQ ID NO. 13-SEQ ID NO. 24;
a primer for detecting vibrio vulnificus, which is shown as SEQ ID NO. 25-SEQ ID NO. 36;
primers for detecting aeromonas with sequences shown as SEQ ID NO. 37-SEQ ID NO. 46;
primers for detecting leptospira with sequences shown as SEQ ID NO. 47-SEQ ID NO. 56;
primers for detecting leptospira renifolia, which are shown in SEQ ID NO. 57-SEQ ID NO. 68;
primers for detecting hantavirus with sequences shown as SEQ ID NO. 69-SEQ ID NO. 74;
primers for detecting the Hancheng virus with the sequences shown as SEQ ID NO. 75-SEQ ID NO. 88;
primers for detecting California encephalitis virus, the sequences of which are shown as SEQ ID NO. 89-SEQ ID NO. 100;
primers for detecting the rift valley fever virus, the sequences of which are shown as SEQ ID NO. 101-SEQ ID NO. 110;
primers for detecting Crimedes-Congo hemorrhagic fever viruses, with the sequences shown in SEQ ID No. 111-SEQ ID No. 118;
a primer for detecting the novel bunyavirus, the sequence of which is shown as SEQ ID NO. 119-SEQ ID NO. 126;
primers for detecting yellow fever virus with the sequences shown as SEQ ID NO. 127-SEQ ID NO. 134;
primers for detecting dengue virus, the sequences of which are shown as SEQ ID NO. 135-SEQ ID NO. 140;
a primer for detecting Japanese encephalitis virus, the sequence of which is shown as SEQ ID NO. 141-SEQ ID NO. 150;
primers for detecting West Nile virus with sequences shown as SEQ ID NO. 151-SEQ ID NO. 158;
primers for detecting St.Louis encephalitis virus with sequences shown in SEQ ID NO. 159-SEQ ID NO. 166;
primers for detecting the ursavirus shown in SEQ ID NO. 167-SEQ ID NO. 174;
primers for detecting the forest encephalitis virus, the sequences of which are shown as SEQ ID NO. 175-SEQ ID NO. 180;
primers for detecting Zika virus, the sequences of which are shown as SEQ ID NO. 181-192;
primers for detecting ALKhurma hemorrhagic fever virus with the sequence shown in SEQ ID NO. 193-SEQ ID NO. 200;
primers for detecting lymphocytic choriomeningitis viruses, the sequences of which are shown as SEQ ID NO. 201-SEQ ID NO. 208;
primers for detecting the Laxavirus, the sequences of which are shown as SEQ ID NO. 209-SEQ ID NO. 218;
primers for detecting the dove-tail virus, the sequences of which are shown as SEQ ID NO. 219-SEQ ID NO. 226;
primers for detecting the melon-Rauvolfia with the sequences shown as SEQ ID NO. 227-SEQ ID NO. 234;
primers for detecting equine autumn wave virus, the sequences of which are shown as SEQ ID NO. 235-SEQ ID NO. 242;
primers for detecting Ebola virus, the sequences of which are shown as SEQ ID NO. 243-SEQ ID NO. 246;
the sequence is shown as SEQ ID NO. 247-SEQ ID NO. 254.
The sequence is shown as SEQ ID NO. 255-SEQ ID NO.262 and is used for detecting the primer of the Casinolin disease virus.
2. The use of the primer for detecting a causative agent of pyretic accompanying hemorrhage as defined in claim 1, for preparing a kit for detecting causative agent of pyretic accompanying hemorrhage.
3. A kit for detecting a causative agent of fever with bleeding, the kit comprising the primer for detecting a causative agent of fever with bleeding of claim 1.
4. The kit for detecting a causative agent of fever with bleeding according to claim 3, wherein the kit further comprises Multi-pcr buffer and RT Enzyme.
5. A method of detecting a causative agent of fever with bleeding, comprising the steps of: multiplex PCR amplification is performed using cDNA of a sample to be tested as a template and the primer for detecting a causative agent of fever with bleeding as set forth in claim 1.
6. The method for detecting a hemorrhagic disease with fever according to claim 5, wherein the working concentrations of the primers for detecting yersinia pestis, streptococcus suis, vibrio vulnificus, aeromonas, dacarbandimonas, aeromonas hydrophila are 180nM to 220nM; the working concentration of the primer for detecting leptospira wild and leptospira kidney is 230 nM-280 nM; the working concentration of the primer for detecting hantavirus, hancheng virus, california encephalitis virus, rift valley fever virus, crimiya-Congo hemorrhagic fever virus, novel bunyavirus, yellow fever virus, dengue fever virus, japanese encephalitis virus, west Nile virus, st.Louis encephalitis virus, wusu graph virus, forest encephalitis virus, zika virus, ALKhurma hemorrhagic fever virus, lymphocytic choriomeningitis virus, lassa virus, dove, guanaratovirus, markov virus, ebola virus and sand fly virus is 380 nM-420 nM.
7. The method for detecting a causative agent of fever with bleeding according to claim 5, wherein the cDNA is obtained by reverse transcription using RNA of a sample to be detected as a template, and the reaction system of the reverse transcription is: template RNA 14. Mu.l, random hexamers 2. Mu.l, 10 XRT Mix 2. Mu.l, reverse transcriptase 2. Mu.l; the reaction procedure of reverse transcription is as follows: 25 ℃ for 5min,37 ℃ for 45min and 85 ℃ for 5s.
8. The method of claim 5, wherein the multiplex PCR amplified reaction system comprises: 5x Multi-PCRbuffer 5. Mu.l, RT Enzyme Mix 3. Mu.l, primer pool 7.5. Mu.l, cDNA9.5. Mu.l.
9. The method for detecting a causative agent of fever with bleeding according to any one of claims 5 to 8, wherein the reaction procedure of multiplex PCR amplification is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 60℃for 1min, extension at 72℃for 1min,28 cycles; after the cycle is completed, the extension is carried out for 1min at 72 ℃.
10. The method of detecting a bleeding-causing pathogen according to any one of claims 5 to 8, wherein the multiplex PCR amplification further comprises a second round of PCR amplification, and the reaction system of the second round of PCR amplification comprises: PCRmix 25. Mu.l, index N52.5. Mu.l, index N72.5. Mu.l, multiplex PCR amplification product 20. Mu.l; the reaction procedure of the second round of PCR amplification is as follows: 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 60℃for 15s, extension at 72℃for 30s,10cyc; and after the circulation is finished, the temperature is 72 ℃ and the extension is carried out for 5min.
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