CN112094930B - Streptococcus pneumoniae serum typing kit and typing method - Google Patents
Streptococcus pneumoniae serum typing kit and typing method Download PDFInfo
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Abstract
The invention discloses a streptococcus pneumoniae serotyping kit and a typing method, and streptococcus pneumoniae serotyping primers and probe sequences are shown in SEQ ID No. 1-183. The streptococcus pneumoniae serotyping kit provided by the invention can realize two-tube simultaneous typing of 92 streptococcus pneumoniae serotypes based on a fusion array technology, and has the advantages of wide coverage serotypes, simplicity and convenience in operation, low cost, high specificity and rapid detection.
Description
Technical Field
The invention relates to a streptococcus pneumoniae serotyping kit and a method.
Background
Streptococcus pneumoniae (Streptococcus pneumoniae) belongs to the genus Streptococcus and is the earliest recognized gram-positive bacterium in humans, and diseases caused by Streptococcus pneumoniae can be divided into Invasive Pneumococcal Disease (IPD) and non-invasive pneumococcal disease (non-IPD, NIPD). Pneumococcal disease is the leading cause of death in children under five years of age and is one of the serious public health problems worldwide.
Streptococcus pneumoniae is not a single bacterium, and its strong pathogenic and colonising capacity is associated with the actual presence of over 90 serotypes. The capsular polysaccharide of the outermost layer of streptococcus pneumoniae is the most important virulence factor, and the pathogenicity of streptococcus pneumoniae is closely related to the capsular polysaccharide of streptococcus pneumoniae. The capsular polysaccharide structures of different serotypes of streptococcus pneumoniae differ, and thus the polysaccharide capsule determines the serotype specificity thereof. To date, streptococcus pneumoniae has been divided into 46 serogroups, 92 serotypes, using a numerical designation for serogroups such as 6, depending on the structure of the polysaccharide capsule, with multiple serotypes in a group being distinguished by the addition of letters such as 6A-6D. The viability and virulence of streptococcus pneumoniae varies from capsular serotype to capsular serotype.
As the phenomenon of drug resistance of streptococcus pneumoniae has become more severe with the use of antibiotics worldwide, streptococcus pneumoniae vaccines are now an effective means of preventing streptococcus pneumoniae infection. WHO ranks the streptococcus pneumoniae disease in children as a disease that requires a very high priority for vaccine prevention. And the development of streptococcus pneumoniae vaccines is mostly based on the streptococcus pneumoniae polysaccharide capsule. Currently, pneumococcal vaccines approved for marketing worldwide include the serotype classes: the vaccine comprises 23-valent polysaccharide vaccine (PPV 23) for children over 2 years old and the elderly, and 7-valent (PCV 7), 10-valent (PCV 10) and 13-valent (PCV 13) conjugate vaccine for children under 2 years old.
Monitoring studies have shown that the incidence of pneumococcal disease and the prevalence of vaccine serotypes are significantly reduced after the initial application of PCV7 vaccine, but with the widespread application of vaccines, there is an increase in non-vaccine serotypes of streptococcus pneumoniae to varying degrees around the world, a phenomenon known as serotype replacement. Currently, PCV13 is on the market and serotype replacement studies are of great interest.
At present, a rapid and clinically applicable serotype typing method is urgently needed for monitoring the serotype replacement situation of streptococcus pneumoniae, analyzing the serotype replacement trend and understanding the domestic streptococcus pneumoniae serotype prevalence situation, so that the application effect of the streptococcus pneumoniae vaccine can be evaluated, and a basis is provided for developing a wide-range and effective vaccine. There are two main current methods for serotyping streptococcus pneumoniae: capsular swelling method and genotype detection method.
Capsular swelling experiments judge serotypes based on the principle of observing whether specific serum and structures such as lipopolysaccharide, flagellar antigen or capsular polysaccharide of bacteria have agglutination reactions or not, and perform serogrouping and typing on streptococcus pneumoniae, which is still the gold standard for detecting streptococcus pneumoniae serotypes at present, but the wide requirements of demand areas for serotype detection in disease diagnosis, epidemiological investigation and the like in actual work cannot be met due to the defects that rabbit antiserum is various in types, expensive in price, complex in operation, and large in artificial error due to the fact that results are mainly read by naked eyes. In addition, recent research shows that the phenomenon of common carrying of multiple serotypes exists widely in the population, but the capsular swelling test can only identify the dominant serotype and cannot simultaneously identify mixed serotypes.
In recent years, molecular biological assays have become the mainstream of the serotyping technique for streptococcus pneumoniae. The streptococcus pneumoniae molecular typing detection method based on serotype specific genes is developed rapidly due to the advantages of rapidness, good specificity, high sensitivity and strong resolution, and samples for detection do not need to be cultured by live bacteria. There are studies that show that the capsular synthesis of almost all serotypes of streptococcus pneumoniae is controlled by its capsular polysaccharide gene cluster (cps), which includes mainly wzx and wzy genes. The sequence of each serotype is different from two bases to completely different, the high heterogeneity of the capsular operons among different serotypes is the theoretical basis of molecular typing of the serotypes, and designing corresponding primers aiming at specific genes of each serotype is the most direct and effective molecular typing method, and various molecular methods are continuously explored. At present, molecular typing technologies of Spn mainly include mPCR and fluorescent quantitative PCR (qPCR), a gene chip method, high-throughput Whole Genome Sequencing (WGS), etc., but because RT-PCR covers less serum, the multiplex PCR operation is more cumbersome, and cross contamination easily occurs; the gene chip technology and the high-throughput whole genome sequencing have the defects of long time consumption, high price and the like, and can not meet the requirements of disease diagnosis, epidemiological investigation and the like in actual work.
At present, related streptococcus pneumoniae serotyping products exist in the market at home, and common streptococcus pneumoniae typing kits mainly comprise the following components:
(1) the Danish SSI pneumococcal serum latex agglutination typing kit judges the serotype based on the principle of observing whether the specific serum and structures of lipopolysaccharide, flagellar antigen or capsular polysaccharide of bacteria have agglutination reaction or not through a latex agglutination test, the kit needs to use 14 bottles of latex reagents to perform serological grouping and typing on SP, but the rabbit antiserum has the defects of various types, high price, complex operation, results mainly judged by naked eyes, large manual error and the like, and the kit can only identify the dominant serotype and cannot identify various serotypes simultaneously, so that the kit cannot meet the wide requirements on serotype detection and serotype replacement condition monitoring in actual work.
(2) The pneumococcal serotyping kit based on the real-time PCR technology is used for typing streptococcus pneumoniae serotypes based on the real-time PCR technology, fluorescent groups are added into a PCR reaction system, real-time monitoring is carried out by utilizing fluorescent group signals, quantitative and qualitative analysis can be carried out on initial model streptococcus pneumoniae templates, the operation is simple, the cost is low, but the reagent kit covers few serotypes (covers 23 common serotypes), and most of the uncommon serotypes cannot be detected.
(3) The reagent kit is used for typing streptococcus pneumoniae serotypes based on a gene chip, specific primers are used for amplifying and marking genome DNA of a sample to be detected, the sample to be detected is hybridized with oligonucleotide probes anchored on the chip, and different serotypes of streptococcus pneumoniae can be detected according to hybridization signals. The hybridization process of the detection probe of the sample to be detected needs uncovering treatment after PCR, cross contamination is easy to occur, and only one sample to be detected can be detected by one gene chip, so the reagent is relatively expensive.
As can be known from query and market research, the related products in the market have the defects of complex operation, long time consumption, less coverage serotype, high cost and the like, and can not meet the requirements of actual typing work.
Disclosure of Invention
Based on the problems, the invention establishes a streptococcus pneumoniae serotyping kit capable of overcoming the limitation of the existing molecular biology technology, and the streptococcus pneumoniae serotyping kit comprises a nucleic acid sequence shown as SEQ ID NO: 1-SEQ ID NO: 183, respectively, and a sequence shown in 183. The method avoids PCR post-treatment while realizing the typing of 92 kinds of serum of streptococcus pneumoniae, has simple, convenient and quick operation and low cost, ensures high sensitivity and high specificity of a system, does not need special equipment, and is suitable for actual typing work.
The streptococcus pneumoniae typing kit provided by the invention can realize two-tube five-channel detection of 92 streptococcus pneumoniae serotypes based on a fusion array (MeltArray) technology, wherein a tube A detects 50 serotypes, a tube B detects 42 serotypes, and the distribution of various streptococcus pneumoniae serotypes is shown in a table 1, so that the streptococcus pneumoniae typing kit is suitable for monitoring serotype replacement in actual work.
TABLE 1A, B tube serotype distribution
Advantageous effects
(1) The primer is a detection primer which is screened out through optimization and secondary screening of a large number of tests and has high efficiency and stable amplification. Through the specific sequence design of the primers and the addition of a section of universal primer sequence at the 5' end of the specific primer, the detection sensitivity (50copes) and specificity of the primers are improved.
(2) The medium probe of the invention is also a detection probe with strong specificity screened out through the optimization of a large number of tests. The detection specificity and accuracy of the medium probe are improved by the specific sequence design of the probe and the adjustment of the length and the position of the probe;
(3) the invention provides a kit for typing all serotypes of streptococcus pneumoniae, and establishes a method for detecting nucleic acid based on a fusion array technology, so that a plurality of target sequences can be detected simultaneously in a single reaction tube. The invention can carry out typing on 92 streptococcus pneumoniae serums in one test, has large serum typing flux, can accurately determine which serotype the streptococcus pneumoniae is, improves the typing efficiency and further reduces the cost.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a graph of the serotyping results of five-channel 50 Streptococcus pneumoniae strains in tube A according to the present invention. For all serotypes covered by the invention, characteristic melting peaks can be detected without any non-specific cross signals.
Panel A is a typical result graph of six detection serotype (15B/C,20,9N/9L,8,19F,3) plasmids of FAM channel after tube A is combined
B is a typical result chart of plasmids of six detection serotypes (15A/15F,2,22A/22F,5,11A/11D/11F and lytA) of CY5 channel A
The C picture is a typical result picture of six detection serotype (33A/33F/37,29,4,1,35F/47F,6A/6B) plasmids of the HEX channel of the A tube
FIG. D is a typical result chart of six tested serotype (17F,14,12A/12B/12D/44/46,19A,23F,7A/7F) plasmids in the tube ROX channel;
FIG. E is a graph showing typical results of plasmids of six tested serotypes (33B/33D,10A/10B/10C/10F,9A/9V, 18A/18B/18C/18F) in the QUASAR channel in the A tube;
FIG. 2 is a graph showing the serotyping results of five-channel 42 Streptococcus pneumoniae strains in tube B according to the present invention. For all serotypes covered by the invention, characteristic melting peaks can be detected without any non-specific cross signals.
Panel A is a typical result graph of six detection serotype (34,7C,31,43,41AF,48) plasmids of FAM channel after tube A combination
B is a typical result chart of six detection serotypes (23B,17A,47A,6C/6D,28A/28F and lytA) plasmids of CY5 channel A
The C picture is a typical result picture of six detection serotype (21,16F,11B/11C,24A/23B/24F,35A/35C/42,13) plasmids of the HEX channel of the A tube
FIG. D is a typical result chart of plasmids of six detection serotypes (16A,32A/32F,27,33C,19B/19C,23A,25A/25F/38) of a tube ROX channel;
FIG. E is a graph showing typical results of plasmids of six test serotypes (39,35B,45 and 36) in the QUASAR channel in the A tube;
FIGS. 3 and 4 are graphs showing the typing results of an isolated sample of serotype 14 Streptococcus pneumoniae, respectively, in which:
the five graphs in FIG. 3 are the results of five-channel assays of tube A FAM, CY5, HEX, ROX, QUASAR
The five graphs in FIG. 4 show the results of five-channel assays of tube B FAM, CY5, HEX, ROX, QUASAR, respectively.
FIG. 5 shows the sensitivity results of tube A for each serotype.
FIG. 6 shows the sensitivity results of each serotype in tube B.
Detailed Description
1. Design of primers and probes
The invention selects the specific sequence of each serotype as an amplification region, designs specific primers and probes, and realizes the typing of 92 streptococcus pneumoniae serotypes by two tubes and five channels on the basis of multiple PCR (polymerase chain reaction) and by combining a fluorescent probe melting curve technology. The two tubes of the system comprise 112 tailed primers, 56 medium probes and 10 universal probes, wherein the tube A comprises 56 tailed primers, 28 medium probes and 10 universal probes. Tube B contains 58 tailed primers, 29 media probes and 10 universal probes. In the establishing process of the invention, in order to ensure the specificity and the amplification efficiency of the multiple PCR amplification, in the establishing process of a single-plex system, 3 pairs of primer pairs are respectively designed for each detection object, and the primer pairs with high amplification efficiency and different detection objects with nearly consistent amplification efficiency are selected as single-plex optimal primer pairs through pairwise cross combination optimization; in the process of establishing the multiplex system, a primer pair which does not produce non-specific amplification and has the best amplification efficiency is selected. Since the multiplex system contains a plurality of primers, a plurality of primer dimers are easily formed, and thus non-specific amplification occurs, which in turn reduces the amplification efficiency. In order to reduce primer dimer and make system detection sensitivity reach single copy level, the invention introduces a Homo-tag assisted-primer dimer-free (HAND) system to carry out multiple equivalent specific amplification, thereby realizing target enrichment. The sequences of the universal primer Tag, the designed primer, the medium probe and the fluorescent probe are shown in the attached table 1.
2. Extraction of DNA template by thermal cracking
Sucking a proper amount of streptococcus pneumoniae bacterial liquid from an LB liquid culture medium, suspending the streptococcus pneumoniae bacterial liquid in an EP tube containing 200 mu L of ultrapure water, repeatedly blowing, sucking and uniformly mixing the streptococcus pneumoniae bacterial liquid, and thermally cracking the streptococcus pneumoniae bacterial liquid for 20min at 99 ℃ and 1000 rpm. Centrifuging at 6000rpm for 5min, and storing 100 μ L of supernatant in a refrigerator at-20 deg.C for subsequent system clinical performance evaluation experiment.
3. Multiplex real-time PCR amplification reaction system
The buffer type, Mg2+ dosage, dNTP and UNG enzyme, primer proportion, primer dosage, probe dosage, DNA polymerase and the like in the PCR reaction are optimized, and the finally determined multiplex real-time PCR reaction system is shown in Table 2.
TABLE 2 component content of multiplex real-time PCR
| PCR reaction solution Components | Dosage (mu L) | 1 part by weight |
| ddH2O | Completion of the |
|
| 10×Taq01 buffer | 2.5 | 1× |
| 25mM MgCl2 | 7.0 | 7mM |
| 25mM dNTP：dUTP | 0.5 | 0.4mM |
| 100μM Tag | 0.3 | 1.2μM |
| 50 μ M fluorescent probe | 0.02 | 0.04μM |
| 50 mu M tailed primer | 0.02-0.04 | 0.04-0.08 |
| 50 μ M media probe | 0.1-0.2 | 0.2-0.4μM |
| 6U/. mu.L Taq01 UNG enzyme (20:1) | 0.80 | 4.0U |
| Total amount of | 20 |
Note: the treated samples were added to the reaction tube in an amount of 5. mu.L, positive control in an amount of 5. mu.L, and negative control (TE solution or purified water) in an amount of 5. mu.L, respectively, and the total reaction volume was 25. mu.L.
4. Condition setting for multiplex real-time PCR amplification
The finally determined multiplex real-time PCR amplification reaction conditions are shown in Table 3 after comparative optimization of a large number of experiments.
TABLE 3 amplification procedure for multiplex real-time PCR
[ T corresponding to detection object ]mValue ]
The invention uses two tubes and five channels, and simultaneously carries out typing on 92 serotypes.
The A tube detects 50 serotypes and a streptococcus pneumoniae specific gene lytA altogether, the FAM channel detects 8 streptococcus pneumoniae serotypes, 6 vector probes and two fluorescent probes are used, wherein three vector probes share one fluorescent probe and generate three different melting points from low to high, and the other three vector probes share the other fluorescent probe and generate two different melting points from low to high. The Cy5 channel is used for detecting 9 streptococcus pneumoniae serotypes and a streptococcus pneumoniae specific gene lytA, 6 vector sub-probes and two fluorescent probes are used, wherein three vector sub-probes share one fluorescent probe and generate three different melting points from low to high, the other three vector sub-probes share the other fluorescent probe and generate two different melting points from low to high. The HEX channel is used for detecting 10 streptococcus pneumoniae serotypes, 6 medium sub-probes and two fluorescent probes are used, wherein three medium sub-probes share one fluorescent probe and generate two different melting points from low to high, the other three medium sub-probes share the other fluorescent probe, and three different melting points are generated from low to high. The ROX channel is used for detecting 11 streptococcus pneumoniae serotypes, 6 medium sub-probes and two fluorescent probes are used, wherein three medium sub-probes share one fluorescent probe and generate two different melting points from low to high, the other three medium sub-probes share the other fluorescent probe, and three different melting points are generated from low to high. The QUASAR channel detects 12 streptococcus pneumoniae serotypes, 4 medium sub-probes and two fluorescent probes are used, wherein the three medium sub-probes share one fluorescent probe and generate two different melting points from low to high, and the other medium sub-probe generates one melting point by using the other fluorescent probe. A pipe T corresponding to each parting objectmThe values are shown in Table 5.
B tube detects 42 serotypes and one streptococcus pneumoniae specific gene lytA, and FAM channel detects 9 streptococcus pneumoniae seraThe type of the probe is that 6 medium sub-probes and two fluorescent probes are used, wherein three medium probes share one fluorescent probe and generate three different melting points from low to high, and the other three medium probes share the other fluorescent probe and generate two different melting points from low to high. The Cy5 channel is used for detecting 7 streptococcus pneumoniae serotypes and a streptococcus pneumoniae specific gene lytA, 6 vector sub-probes and two fluorescent probes are used, wherein three vector sub-probes share one fluorescent probe and generate three different melting points from low to high, the other three vector sub-probes share the other fluorescent probe and generate two different melting points from low to high. The HEX channel detects 11 streptococcus pneumoniae serotypes, 6 medium sub-probes and two fluorescent probes are used, wherein three medium sub-probes share one fluorescent probe and generate two different melting points from low to high, the other three medium sub-probes share the other fluorescent probe and generate three different melting points from low to high. The ROX channel is used for detecting 11 streptococcus pneumoniae serotypes, 6 medium sub-probes and two fluorescent probes are used, wherein three medium sub-probes share one fluorescent probe and generate two different melting points from low to high, the other three medium sub-probes share the other fluorescent probe, and three different melting points are generated from low to high. The QUASAR channel detects 4 streptococcus pneumoniae serotypes, 4 medium sub-probes and two fluorescent probes are used, wherein the three medium sub-probes share one fluorescent probe and generate two different melting points from low to high, and the other medium sub-probe generates one melting point by using the other fluorescent probe. B is used for managing T corresponding to each parting objectmThe values are shown in Table 5.
TABLE 4A tube T for each typing objectmValue of
TABLE 5B tubes T for each typing objectmValue of
TABLE 6A detection primer probes corresponding to respective typing targets
TABLE 7B tubes detection primer probes corresponding to each typing object
The established streptococcus pneumoniae typing kit is used for typing streptococcus pneumoniae, the typing result accuracy is 100%, and the specificity is 100%.
Referring to fig. 5 and 6, the minimum detection limit for each serotype was 50 copies/reaction, and the system sensitivity was 50 copies/reaction.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
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<213> Artificial Sequence (Artificial Sequence)
<400> 32
ccggcgggga gggaccgtcg tcccaggacc agcagctcac caccccgccg g 51
<210> 33
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gcaagccctc acgtagcgaa gtagcggatt gctgatttac a 41
<210> 34
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gcaagccctc acgtagcgaa gtccaaagtc agactagatg gt 42
<210> 35
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ggtggtgagc tatgccccaa agacgtctcc tgaaacca 38
<210> 36
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gcaagccctc acgtagcgaa tctactttag cttctcgwtt atgga 45
<210> 37
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcaagccctc acgtagcgaa caacaccgat tgacccact 39
<210> 38
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ggtcctgcca cgattgtagc ccaacaccga ttgacccact gcaggagg 48
<210> 39
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gcaagccctc acgtagcgaa gaagagttca tgacggacta c 41
<210> 40
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gcaagccctc acgtagcgaa gaagagttca tgacggacta c 41
<210> 41
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ggacgacggt cggcaaacct gcttcatctg ctagattgcg t 41
<210> 42
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
atcgccataa aagatagtgc actgagtctc agtccggcga t 41
<210> 43
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcaagccctc acgtagcgaa tctctcctat tttggcttat tggta 45
<210> 44
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gcaagccctc acgtagcgaa tgagtccaag ttgtgaaatt ccat 44
<210> 45
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
cactatcttt tatccacata ggtgcttcag caaaaatccc aagatagac 49
<210> 46
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gcaagccctc acgtagcgaa ctagcgcaaa gttgggagt 39
<210> 47
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gcaagccctc acgtagcgaa gcgagaatca gtttgtcca 39
<210> 48
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
agtggtctat ctccgtagct atctaggttt cagatatgcc ttgtatcca 49
<210> 49
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gcaagccctc acgtagcgaa gcagttagag cggcgaata 39
<210> 50
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gcaagccctc acgtagcgaa acgctttata actcgggaca 40
<210> 51
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gactctctgg taggagtact tatgcttcct ggtgtgttct ccttgt 46
<210> 52
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gcaagccctc acgtagcgaa ttaagtgggc aagtttcatc t 41
<210> 53
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gcaagccctc acgtagcgaa acatatccct ctcccacaag 40
<210> 54
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gactgactct ctagctgata caaactcacc atctagcaaa tcaaacca 48
<210> 55
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
cgcgcgactg ggcagggaca cgcgtcgtct cggacggctg cgcgcg 46
<210> 56
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gcaagccctc acgtagcgaa tgaaacggat tctcgattaa ca 42
<210> 57
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gcaagccctc acgtagcgaa accatttcca ttccattcaa ct 42
<210> 58
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cgcgtgtccc tatccgaact tagagaatag ttctgggatc catct 45
<210> 59
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
gcaagccctc acgtagcgaa tctctatggg gtgaatgata ct 42
<210> 60
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gcaagccctc acgtagcgaa aacccctaaa tctccttgta a 41
<210> 61
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
agccgtccga cttgcaaaaa tctttcttcg acgttattat ttctcca 47
<210> 62
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
cgagcaaaaa gaagtgtgtc acctgtcttg agctcg 36
<210> 63
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gcaagccctc acgtagcgaa agcctactat gagttataga cga 43
<210> 64
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gcaagccctc acgtagcgaa aataggagtt cagtgtcaat act 43
<210> 65
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
gacacacttc ttttcatgcc ttgacttaca agcagacctt agtcgaagtg tgag 54
<210> 66
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gcaagccctc acgtagcgaa acgctttaga gtgtatgagg a 41
<210> 67
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gcaagccctc acgtagcgaa cttagtctct cagatgaatc aca 43
<210> 68
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ggtctcacac tggcgcaggt gtcagaattc cctctacagt 40
<210> 69
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
gcaagccctc acgtagcgaa ggtgttggtg ggcattgta 39
<210> 70
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
gcaagccctc acgtagcgaa gattctaata actgaagcgt tgg 43
<210> 71
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
agacacctct cgggttggtc tatatggtct tacctacaaa gaga 44
<210> 72
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
cggcggagtg ggcacggaga gccgtggaca gactggtgcc acgtctcgca gcaggccgcc 60
g 61
<210> 73
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gcaagccctc acgtagcgaa cgctctaaag atttactgct ct 42
<210> 74
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gcaagccctc acgtagcgaa gtcgtagaac gtccagataa g 41
<210> 75
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
gcgctctccg tgtaatggcc tatattcagc aaaaacgaaa agttggct 48
<210> 76
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gcaagccctc acgtagcgaa tagtcatctc tttactggcg tt 42
<210> 77
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gcaagccctc acgtagcgaa ccaactaacc caacataacc at 42
<210> 78
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gtctgtccag cgctgtccat aacccttcgt cgtatttcca aaggctggac a 51
<210> 79
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
gcaagccctc acgtagcgaa tgggagagat tttcctgaag ca 42
<210> 80
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gcaagccctc acgtagcgaa tggagtctct gtataagatt ggac 44
<210> 81
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
caccacactg tcctaatcca aggttttgaa acatggcttc ttccagaaag 50
<210> 82
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
atcgccaaga atagacagag agagtctacc ctgacaggcg at 42
<210> 83
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gcaagccctc acgtagcgaa tgactactag tcgttggatg aca 43
<210> 84
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gcaagccctc acgtagcgaa tccacaaact tgccttcca 39
<210> 85
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ctctgtctat tctaggagca ataaagagag tcctaagctc aa 42
<210> 86
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gcaagccctc acgtagcgaa gaatcgaatc attccagcaa gt 42
<210> 87
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gcaagccctc acgtagcgaa tgcttcctgg ataccacga 39
<210> 88
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gactctgact gctgtgtcgt ccgtacttcc atcattcaca 40
<210> 89
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gcaagccctc acgtagcgaa gatatacttg ctcgaacggg ta 42
<210> 90
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
gcaagccctc acgtagcgaa tacagatgta gcctccatcg ta 42
<210> 91
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
agggtactct cttgctcgaa cgggtatatt tccgggta 38
<210> 92
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
cgcgccaggc acgaggacag gagagcacgg cacccaggag ggggcgcg 48
<210> 93
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
gcaagccctc acgtagcgaa ggaagggagt tgaatcaacc ta 42
<210> 94
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gcaagccctc acgtagcgaa tcctacaaat cctatctcaa tgtagt 46
<210> 95
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ggtgccgtgc tcatccattt tgttacaaac cctatccctc tcc 43
<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gcaagccctc acgtagcgaa 20
<210> 97
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gcaagccctc acgtagcgaa ccttattgtt gtagtggcag t 41
<210> 98
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
gcaagccctc acgtagcgaa ggcaatccat tggtactctt ca 42
<210> 99
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
cttactgttc tcaagatggg attattgaag ctcttaatcg agaact 46
<210> 100
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
gcaagccctc acgtagcgaa tgagaataga ggatatggag ca 42
<210> 101
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
gcaagccctc acgtagcgaa gacccaccaa ccatagca 38
<210> 102
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
ctctgatact gtccagatat agtcattccc aatcaggaag tga 43
<210> 103
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
gcaagccctc acgtagcgaa gtatcgcgtt gttgttctg 39
<210> 104
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
gcaagccctc acgtagcgaa catctgagaa atgctacgac ca 42
<210> 105
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
gctgtgactg acctctctat tatcatctga gaaatgctac gaccaga 47
<210> 106
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gcgcgccagc ggacgaggct gaccaccgca cggaggtgcc ggcgcgc 47
<210> 107
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
gcaagccctc acgtagcgaa ttcttatcgc ttccattgtc ag 42
<210> 108
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
gcaagccctc acgtagcgaa cgctcctgaa gtcactg 37
<210> 109
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
gtcagcctcg tgcattccta taacagcacc taaatatcct tcccca 46
<210> 110
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gcaagccctc acgtagcgaa gagttttaaa gtcggaacac ct 42
<210> 111
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
gcaagccctc acgtagcgaa cacacataca tatctcaacc att 43
<210> 112
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
ctccggtgca cgaagttatt agaggatgct ctaaacttga tctcaa 46
<210> 113
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
gcaagccctc acgtagcgaa tcgatcagtg gcatttggag t 41
<210> 114
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
gcaagccctc acgtagcgaa taggtgtttc caaatcaccg a 41
<210> 115
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ggcacctccg agcaccatta cctacaaaaa taatcgacgg aatca 45
<210> 116
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
gcaagccctc acgtagcgaa ttgatagatc gttacagtgg gaa 43
<210> 117
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gcaagccctc acgtagcgaa tgtggaatct tataagcaac tgaa 44
<210> 118
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
gttcgttgag ttttaagttg gtgggttaat aacacttcag ataggt 46
<210> 119
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
gcaagccctc acgtagcgaa gagcctattg tggtacagtt ac 42
<210> 120
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
gcaagccctc acgtagcgaa tttccaagca atcgaatgac a 41
<210> 121
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gtcatcgttg agtcttggtt acgtaagatt atttcgccaa agt 43
<210> 122
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gcaagccctc acgtagcgaa tcgaactgga gggttagtaa ga 42
<210> 123
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gcaagccctc acgtagcgaa tgttctagag tccgttatgg ca 42
<210> 124
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
gatccacatc gggttagtaa gaggatcatt agggtttgtc catccga 47
<210> 125
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
gcaagccctc acgtagcgaa tagtaggcag gttagcttct 40
<210> 126
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gcaagccctc acgtagcgaa tcgttaaaat cttcatcaat ctct 44
<210> 127
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
ggacgacggt aaggggtatt tttaaagatt gagccgcttc atattcaga 49
<210> 128
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gcaagccctc acgtagcgaa cattgatatt agcaggtgta ggt 43
<210> 129
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
gcaagccctc acgtagcgaa gtatgacccc actaacacca 40
<210> 130
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
ggtcctgcca cagagtacat ttcatatcca cgccaattcc atatgagcag gagg 54
<210> 131
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gcaagccctc acgtagcgaa gaagagttca tgacggacta c 41
<210> 132
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
gcaagccctc acgtagcgaa gaagagttca tgacggacta c 41
<210> 133
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
ggtggtgagc tacgcaatct agcagatgaa gcaggtttg 39
<210> 134
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
gcaagccctc acgtagcgaa ctattgtcag tgtcggattg aa 42
<210> 135
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
gcaagccctc acgtagcgaa gtgaaacatg aacaacggac t 41
<210> 136
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
cactatcttt tatctgtcaa cacagacaca atcgctgcc 39
<210> 137
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
gcaagccctc acgtagcgaa gcgtgggtgt taaaagatca 40
<210> 138
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
gcaagccctc acgtagcgaa agcactacgg ctaaatgtaa g 41
<210> 139
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
agtggtctat cttgctagta actcgttgtt gaccgaaaaa accacca 47
<210> 140
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
gcaagccctc acgtagcgaa taaccttttt ggggcatgt 39
<210> 141
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
caagccctca cgtagcgaat ccctctcttc caaaatactc t 41
<210> 142
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gactctctgg tgagcacagt caaaatttgt aataggaagg ct 42
<210> 143
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
gcaagccctc acgtagcgaa gacgtagcca aagtctcctt 40
<210> 144
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gcaagccctc acgtagcgaa ctctggtagg cgaaatctc 39
<210> 145
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
cgcgtgtccc taccaagctt actactcaat actactagga gca 43
<210> 146
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
gcaagccctc acgtagcgaa tcccaatcat tacaactccc tat 43
<210> 147
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
gcaagccctc acgtagcgaa cacacaattg cagggagtag a 41
<210> 148
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
gggagacgag ccagagatta ccttagtacc atagttagca actcct 46
<210> 149
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
gcaagccctc acgtagcgaa tgtaatgtgg ttttcaggac t 41
<210> 150
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gcaagccctc acgtagcgaa cagcagaaat agtacgaagg a 41
<210> 151
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
gactgactct ctctctttaa agattacaat agcagggagc cc 42
<210> 152
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
caagccctca cgtagcgaat gactcttata tattcttcaa ggga 44
<210> 153
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
gcaagccctc acgtagcgaa agcggatcac accttctg 38
<210> 154
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
gacacacttc ttttacaatg ttctgccaga ttggagttgt aggaactgt 49
<210> 155
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gcaagccctc acgtagcgaa tgtcttagca attgtgtttg tct 43
<210> 156
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
gcaagccctc acgtagcgaa gcagcaatat cggatccat 39
<210> 157
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
ggtctcacac tactccaagg ctcacattat ttaaatacgg tatgctata 49
<210> 158
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
gcaagccctc acgtagcgaa gtatctgatt ggtgtcgaga gt 42
<210> 159
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
gcaagccctc acgtagcgaa aagatactgc tccagctca 39
<210> 160
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
agacacctct ccccatattt ctcgctacgc taacacct 38
<210> 161
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
gcaagccctc acgtagcgaa gagtgctact ggtttctgt 39
<210> 162
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
gcaagccctc acgtagcgaa tcaactccca gtatctaaat cct 43
<210> 163
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
gagtccagcg ctccaatcca cctccataaa cgaatgct 38
<210> 164
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
gcaagccctc acgtagcgaa ggcaggtata agtattatcg ggt 43
<210> 165
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
gcaagccctc acgtagcgaa tccacaaact tgtcttccat act 43
<210> 166
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
cggctctccg tccctgtcac agtaggttta ggcttctct 39
<210> 167
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
gcaagccctc acgtagcgaa tggcgattga gaatagggt 39
<210> 168
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
gcaagccctc acgtagcgaa cgctgcttta cttacactga g 41
<210> 169
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
ggacactgtc cactcttctg aattaattgg cggtaaacaa ttaaggcgt 49
<210> 170
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gcaagccctc acgtagcgaa agttattttg gggtgcgtt 39
<210> 171
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
gcaagccctc acgtagcgaa ttgctccaag tttcctcaga 40
<210> 172
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
cagggtccac actcatcgca aaatacccag atgtactggt ga 42
<210> 173
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
gcaagccctc acgtagcgaa ggtttgggaa cttgatgtct aa 42
<210> 174
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
gcaagccctc acgtagcgaa gcccctgcta aatgatacct c 41
<210> 175
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
ctctgtctat tctgactgta aaactaacta gtgaataggt acctgtct 48
<210> 176
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
gcaagccctc acgtagcgaa ggtagtgtaa cgagctatac t 41
<210> 177
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
gcaagccctc acgtagcgaa tgaaaactca atgtaatcag gaa 43
<210> 178
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
gactctgact ggaacaacat agcaacacta atgataaagg ctact 45
<210> 179
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
gcaagccctc acgtagcgaa gcgtttctgt ttcacggat 39
<210> 180
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
gcaagccctc acgtagcgaa ccaagacgag tcgatttc 38
<210> 181
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
agggtactct cgagtcgatt tcgctgtata atacataaca cgact 45
<210> 182
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
gcaagccctc acgtagcgaa gtctattcag cccttctggt t 41
<210> 183
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
gcaagccctc acgtagcgaa ggaactgcac ctaagagaga t 41
<210> 184
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
acccgtgctc aacgtatctc attgtagcgg gcattcttgt act 43
Claims (5)
1. A streptococcus pneumoniae serotyping kit is characterized in that: comprises the amino acid sequence shown as SEQ ID NO: 1-SEQ ID NO: 184: wherein, SEQ ID NO: 1-SEQ ID NO: 95 are arranged in a tube A; SEQ ID NO: 96-SEQ ID NO: 184, and the sequence shown in SEQ ID NO: 2. 22, 32, 42, 52, 62, 72, 82 and 92.
2. The streptococcus pneumoniae serotyping kit according to claim 1, wherein: also includes Taq01 buffer, MgCl2And dNTP: dUTP, Taq01, and UNG enzyme.
3. A multiplex PCR reaction system for serotyping Streptococcus pneumoniae, comprising:
mu L of component usage amount of PCR reaction solution
Completion of ddH2O
10× Taq01 buffer 2.5
25 mM MgCl2 7.0
25 mM dNTP：dUTP 0.5
100 µM Tag 0.3
50 mu M fluorescent probe 0.02
0.02-0.04 mu M tailing primer
0.1-0.2 mu M medium probe
6U/muL Taq01 UNG enzyme =20: 10.80
Total 20
5ul of sample is added into the PCR reaction solution, and the total reaction volume is 25 ul;
the reaction conditions are as follows: 2min at 50 ℃; 5min at 95 ℃; 50 cycles of 95 ℃ for 20s and 60 ℃ for 1 min; 20min at 35 ℃; 2min at 95 ℃; 2min at 45 ℃;
wherein the sequences of the fluorescent probe, the tailing primer and the medium probe are shown as SEQ ID NO: 1-SEQ ID NO: 184.
4. A method of serotyping streptococcus pneumoniae bacteria for non-disease diagnostic and therapeutic purposes comprising the steps of:
1) designing a universal primer Tag, a designed primer, a medium probe and a fluorescent probe sequence, wherein the sequences are shown as SEQ ID NO: 1-SEQ ID NO: 184; and divided into two tubes, SEQ ID NO: 1-SEQ ID NO: 95 are arranged in a tube A; the amino acid sequence of SEQ ID NO: 96-SEQ ID NO: 184 is arranged in the tube B; and also included in tube B is SEQ ID NO: 2. 22, 32, 42, 52, 62, 72, 82, and 92;
2) extracting DNA of streptococcus pneumoniae;
3) adopting multiplex real-time PCR amplification, wherein the PCR reaction system is as follows:
mu L of component usage amount of PCR reaction solution
Completion of ddH2O
10× Taq01 buffer 2.5
25 mM MgCl2 7.0
25 mM dNTP：dUTP 0.5
100 µM Tag 0.3
50 mu M fluorescent probe 0.02
0.02-0.04 mu M tailing primer
0.1-0.2 mu M medium probe
6U/muL Taq01 UNG enzyme =20: 10.80
Total 20 of
Respectively adding 5 muL of treated samples, 5 muL of positive quality control products, 5 muL of negative quality control products and 25 muL of total reaction volume into the reaction tube;
the reaction conditions are as follows: 2min at 50 ℃; 5min at 95 ℃; 50 cycles of 95 ℃ for 20s and 60 ℃ for 1 min; 20min at 35 ℃; 2min at 95 ℃; 2min at 45 ℃; the PCR reaction instrument is finally heated from 45 ℃ to 95 ℃ at a heating rate of 0.04 ℃/s, and simultaneously, the fluorescence signals of a plurality of channels are collected.
5. The method of serotyping Streptococcus pneumoniae according to claim 4, wherein: the multiple channels are five channels of FAM, HEX, ROX, CY5 and QUASAR.
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