CN111534611A - Fluorescent quantitative PCR method for detecting toxigenic streptococcus suis and corresponding kit - Google Patents
Fluorescent quantitative PCR method for detecting toxigenic streptococcus suis and corresponding kit Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR method for detecting toxigenic streptococcus suis and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the streptococcus suis from other streptococcus and the streptococcus producing poison and non-producing poison, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the toxigenic streptococcus suis has the advantages of high sensitivity, rapidness, convenience, good specificity, rigorous and accurate judgment and the like, and has good application prospect and market value.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a method for carrying out fluorescent quantitative PCR detection on streptococcus suis producing viruses through specific genes and a corresponding detection kit.
Background
Streptococcus suis (Streptococcus suis) is a gram-positive cocci with a membrane. Grow on blood plate culture medium, form the hemolytic ring around the colony. Streptococcus suis can be classified as Lancefield subgroup D streptococci according to its cell wall antigen component.
The streptococcus suis is a zoonosis pathogenic bacterium, and is divided into 35 (types 1-34 and 1/2) serotypes according to the difference of capsular antigens (CPS), wherein the types 1, 2, 7 and 9 are the most pathogenic streptococcus suis. Pathogenic factors of the streptococcus suis comprise capsular polysaccharide, lysozyme releasing protein, extracellular factors and hemolysin. The distribution of serotypes of streptococcus suis and phenotypes of virulence factors are regionally different.
Some serotypes of streptococcus suis are pathogenic and primarily infect through wounds. The natural sites of infection with Streptococcus suis are the upper respiratory tract (especially the tonsils and nasal cavities), the reproductive tract and the digestive tract of pigs. Pigs are susceptible to a high degree in a variety of animals. The diseases of pigs of various ages can cause diseases such as acute septicemia, meningitis, arthritis, endocarditis, pneumonia and the like of the pigs. In human infection, streptococcus suis can infect humans through droplet or wound touch, and diseases such as septicemia, streptococcal infection toxic shock and perceptual deafness are caused. Pathogenic bacteria can invade meninges, and the patients have symptoms of fever, unclear mind, coma and the like. Pathogenic bacteria enter blood, and septicemia occurs, which causes subcutaneous hemorrhage and acute organ failure.
At present, a great deal of research is carried out on streptococcus suis at home and abroad, and a plurality of effective detection methods are established, wherein pure culture, physiological and biochemical detection, serological typing and phage typing of bacteria are widely applied, but the problems of time consumption, low sensitivity and the like still exist. In recent years, with the wide application of high-throughput genome sequencing technology, more and more streptococcus suis strains are subjected to whole genome sequencing, and a reference database is provided for molecular diagnosis technology to lock specific sequences of streptococcus suis. Currently, molecular biology techniques are being applied to the diagnostic detection of streptococcus suis gradually due to the advantages of high sensitivity, high specificity, short experimental period, easy operation and the like.
Disclosure of Invention
One of the purposes of the invention is to provide a fluorescent quantitative PCR method for detecting the toxigenic streptococcus suis.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting a streptococcus suis specific gene rpoB and toxigenic specific genes gene1, gene2, gene3 and gene 4;
s4, reading the Ct value of the amplification; when the Ct values of at least 1 gene in the streptococcus suis specific gene and the toxigenic specific gene are both less than 35, the detection result of the toxigenic streptococcus suis is positive; when the Ct value of the streptococcus suis specific gene rpoB is more than 35 and/or the Ct values of the toxigenic specific genes gene1, gene2, gene3 and gene4 are more than 35, the detection result of the toxigenic streptococcus suis is negative.
As a preferred technical scheme, a detection primer of the streptococcus suis specific gene rpoB (NCBI accession number: DQ232478.1) is shown as SEQ ID NO: 1-2;
SEQ ID NO:1(5'-ACAACTTCGAGGATGCGGTT-3')；
SEQ ID NO:2(5'-CCCATTTCGTCCAAGTTGCG-3')。
the detection primer of the specific gene1 for toxin production (NCBI Accession Number: ADE32332.1) is shown as SEQ ID NO. 3-4;
SEQ ID NO:3(5'-ATGCAGGGCCGAGAAACTC-3')；
SEQ ID NO:4(5'-ACCAATCACCCCAACGTCAT-3')。
the detection primer of the specific gene2 for toxin production (NCBI Accession Number: SQH00723.1) is shown as SEQ ID NO. 5-6;
SEQ ID NO:5(5'-TCGAAGAAGCAGGCTACAAGG-3')；
SEQ ID NO:6(5'-TCAACCAAACCTGCTTCAACTG-3')。
the detection primer of the specific gene3 for toxin production (NCBI Accession Number: ADE31655.1) is shown as SEQ ID NO. 7-8;
SEQ ID NO:7(5'-CCAAACCCTCAAAGCACAGG-3')；
SEQ ID NO:8(5'-TGGGAGCAATGTGGGAGATG-3')。
the detection primer of the specific gene4 for toxin production (NCBI Accession Number: SQH00863.1) is shown as SEQ ID NO. 9-10;
SEQ ID NO:9(5'-ACAAAACACATCGCCCTTGC-3')；
SEQ ID NO:10(5'-TCTCTGGCAACCCGTTCTTC-3')。
another objective of the invention is to provide a fluorescent quantitative PCR kit for detecting the toxigenic Streptococcus suis, which comprises the detection primer of the Streptococcus suis specific gene rpoB and the detection primers of the toxigenic specific genes gene1, gene2, gene3 and gene 4.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, through a large amount of early research data on microorganisms of Streptococcus suis, other Streptococcus species, toxigenic and non-toxigenic Streptococcus species, orphan genes capable of distinguishing the Streptococcus suis from the other Streptococcus species and the toxigenic and non-toxigenic Streptococcus species are excavated, whether the bacteria sample and the environment sample contain the toxigenic Streptococcus suis or not is accurately judged through detection of the specific genes, the early-stage big data mining and the comparison basis among different species are provided, and the selected specific genes have the specificity of the species and the bacteria.
(2) In the invention, by optimizing the design conditions and the experimental conditions, the primers which are optimized for each gene detection amplification condition are selected as the detection primers, so that the amplification efficiency of the primers is improved, the detection primers with stability, high efficiency, high sensitivity and reproducibility can be achieved, and the correct detection of a trace sample is realized.
(3) Compared with other detection methods in the market, the detection method provided by the invention has the advantages that the two-part detection strategy can be used for judging the streptococcus suis, other streptococcus, toxigenic streptococcus and non-toxigenic streptococcus step by step, two detection methods are simultaneously and positively required for judging the streptococcus suis to be toxigenic streptococcus, and the result is more rigorous and accurate.
(4) The invention can realize the detection range from 10 positive bacteria to 10000 positive bacteria, has high sensitivity and wide application range, can detect samples with higher concentration through dilution conversion, has stable detection result and has good reproducibility.
(5) The detection method can be applied to screening and detecting clinical samples from various sources such as tissues, organ lavage fluid, in-vitro secretion, excrement and the like in clinical medicine, accurately judges whether the clinical samples contain the toxigenic streptococcus suis, and is convenient, rapid and sensitive to operate. The detection kit can be applied to the rapid detection of various clinical samples, does not need additional microbial culture, enables gene detection to be applied to daily life, and has the advantages of high sensitivity, strong operability, rapidness, high efficiency and the like.
Drawings
FIG. 1 shows a calibration curve of primers for rpoB gene detection.
Gene1 gene detection primer standard curve of fig. 2.
FIG. 3 is a gene2 gene detection primer standard curve.
FIG. 4 is a gene3 gene detection primer standard curve.
FIG. 5 is a gene4 gene detection primer standard curve.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
The following reagents used in the present invention can be purchased from conventional sources.
TABLE 1
In some embodiments, positive standard plasmids are constructed using the amplification products, and a standard curve of amplification of the primers is detected and plotted to obtain the amplification efficiency of each pair of detection primers, and the amplification efficiency and confidence level of the primer sequences provided by the invention are optimized.
In some embodiments, by detecting specific genes for different positive strains, it is possible to correctly distinguish between Streptococcus suis and other Streptococcus species, toxigenic and non-toxigenic Streptococcus species, and thereby accurately detect and determine toxigenic Streptococcus suis.
In some embodiments, by extracting and detecting 10, 100, 1000 and 10000 positive bacteria, streptococcus suis and other streptococcus species, toxigenic and non-toxigenic streptococcus species can be accurately distinguished, thereby accurately detecting and judging toxigenic streptococcus suis. And can guarantee that as low as 10 positive bacteria can still be accurately detected, and a microbial sample with higher titer can be also accurately detected by diluting to a detection interval.
In some embodiments, the alveolar lavage fluid, the skin secretion, the urine, the vaginal secretion and the filtered water are collected, and the specific genes are detected, so that whether the sample contains the toxigenic streptococcus suis can be quickly and accurately detected, and the convenience and the rapidness are realized.
It will be understood by those skilled in the art that the genes to be detected and the specific primers to be designed in the method can be designed to achieve the corresponding detection purpose through the design of other sections of the same detection object.
The method and the kit for detecting the fluorescent quantitative PCR of the toxigenic streptococcus suis can be widely applied to multiple clinical medical inspection categories such as tissue lavage fluid detection, in-vitro secretion detection, excrement detection and the like in clinical inspection, and can provide a convenient, quick, accurate and efficient gene detection-based monitoring product for clinical medical inspection.
Example 1 Standard Curve for detection primer amplification
1. Cultivation of microorganisms
The alkali peptone water is used as a culture medium of the streptococcus suis, and the alkali peptone water or a flat plate with the pH value of 8.8-9.0 has good growth. The colony diameter on the alkaline plate is 2mm, round, smooth and transparent. And selecting a microorganism sample which is in vigorous growth to perform subsequent experiments.
2. Genomic DNA extraction
Extraction of genomic DNA was performed according to the QIAamp DNA Mini Kit instructions from Qiagen.
3. Standard quality particle preparation of amplification product
DNA extracted from Streptococcus suis was used for amplification with primers for rpoB gene, gene1, gene2, gene3 and gene4, respectively, the amplification products were treated with A, ligated into T-vectors using T4 ligase, and after transduction of competent cells, plasmids were amplified on a large scale. The primer sequences of the 5 genes are shown as SEQ ID NO 1-10, and are specifically shown as table 2.
TABLE 2
4. Plasmid extraction and Standard Curve gradient configuration
Respectively plating standard clonic bacteria of the amplification products of the primers corresponding to the amplification products of rpoB gene, gene1, gene2, gene3 and gene4, collecting the monoclonal bacterial plaques, performing LB culture and shake bacteria overnight for amplification to obtain 5ml of Plasmid positive strains in exponential growth phase, and extracting Plasmid DNA according to the instruction of QIAGEN Plasmid Midi Kit. After extraction, the concentration of plasmid DNA was determined, and by means of 10-fold dilution, 6 standards were prepared with concentration gradients of 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, and 10 pg/. mu.L, respectively.
5. Drawing a standard curve of the detection primer
qPCR amplification of a concentration gradient standard curve was performed according to BioRad iQ SYBR Green SuperMix instructions and the corresponding amplification curves were plotted to obtain the amplification efficiency for each pair of primers. 3 biological replicates and 3 technical replicates were set for each qPCR reaction, with the qPCR reaction system and amplification reaction conditions shown in table 3.
TABLE 3
6. Analysis of results
The result of the detection primer amplification standard curve shows that primers corresponding to rpoB gene, gene1, gene2, gene3 and gene4 are optimized primer design and a reaction system. Linear standard curve confidence R2>0.980, the high amplification efficiency is between 90 and 105 percent, and the primer design and the reaction system are proved to be in the optimal state. The correlation coefficient and the reliability of the amplification curves of 5 genes and the amplification efficiency under 6 concentration gradients are analyzed, the requirements of the optimal state are met, and the 5 pairs of primers can meet the detection requirements under the reaction conditions.
TABLE 4
EXAMPLE 2 Positive and negative sample detection
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
qPCR detection of 3.5 target genes
qPCR amplification of samples was performed as instructed by BioRad iQ SYBR Green SuperMix instructions and Ct value readings were obtained for each pair of primers. 3 biological replicates and 3 technical replicates were set for each qPCR reaction, with the qPCR reaction system and amplification reaction conditions shown in table 5.
TABLE 5
4. Analysis of results
As shown in tables 6 and 7, the results of qPCR amplification with 5 pairs of primers showed that Streptococcus suis exhibited positive results for the rpoB gene, while other Streptococcus showed negative results. Therefore, the detection of rpoB gene in Streptococcus suis and other Streptococcus can be performed to distinguish Streptococcus suis from other Streptococcus better. Meanwhile, the detection results of gene1, gene2, gene3 and gene4 show that the toxigenic streptococcus exhibits positive detection results, while other non-toxigenic streptococcus suis and other streptococcus are negative. Therefore, the detection of gene1, gene2, gene3 and gene4 can better distinguish between toxigenic and non-toxigenic streptococci. In conclusion, the primers corresponding to rpoB gene can satisfy the detection judgment of Streptococcus suis in the genus Streptococcus, and the primers corresponding to gene1, gene2, gene3 and gene4 can satisfy the detection judgment of toxigenic and non-toxigenic Streptococcus.
TABLE 6
TABLE 7
EXAMPLE 3 sensitivity of the detection System
1. Cultivation of microorganisms
The alkali peptone water is used as a culture medium of the streptococcus suis, and the alkali peptone water or a flat plate with the pH value of 8.8-9.0 has good growth. The colony diameter on the alkaline plate is 2mm, round, smooth and transparent. And selecting a microorganism sample which grows vigorously to perform subsequent experiments, collecting thalli, then performing concentration calculation, diluting by 10 to obtain 10 bacteria/ml, 100 bacteria/ml, 1000 bacteria/ml and 10000 bacteria/ml, and performing subsequent DNA extraction experiments on bacterial liquids with different concentration gradients.
2. Genomic DNA extraction
The same as in example 1.
qPCR detection of 3.5 target genes
The same as in example 2.
4. Analysis of results
As shown in tables 8 and 9, the results of qPCR amplification with 5 pairs of primers showed that Streptococcus suis exhibited positive results for rpoB gene in the range from 10 bacteria to 10000 bacteria. Therefore, the detection of rpoB gene can be performed to detect Streptococcus suis in a range of preferably 10 to 10000 bacteria. Meanwhile, in the range from 10 bacteria to 10000 bacteria, the detection results of gene1, gene2, gene3 and gene4 genes show that the toxigenic streptococcus is positive, and other non-toxigenic streptococcus is negative. Therefore, the genes gene1, gene2, gene3 and gene4 can be detected, and the detection and differentiation of the toxigenic and non-toxigenic streptococcus can be well achieved within the range from 10 bacteria to 10000 bacteria. In summary, the detection of the toxigenic streptococcus suis can be well completed through the matching detection of rpoB genes, gene1, gene2, gene3 and gene4, the detection range can be from 10 strains to 10000 strains, and higher strain concentration can be diluted to an optimal detection range through a dilution method, so that expected detection requirements can be met.
TABLE 8
TABLE 9
EXAMPLE 4 detection of clinical samples
1. Clinical sample Collection and extraction of genomic DNA
Different alveolar lavage fluid, skin secretions, urine, vaginal secretions, and filtered water were collected, and genomic DNA was extracted as instructed by QIAamp DNA Mini Kit from Qiagen.
qPCR detection of 2.5 target genes
The same as in example 2.
3. Analysis of results
As shown in tables 10 and 11, the qPCR amplification results for 5 pairs of primers showed that rpoB gene was detected in all samples except one vaginal secretion sample and filtered water, confirming that the corresponding sample source contains streptococcus suis. In addition to an alveolar lavage fluid sample, a urine sample, a skin secretion sample, a vaginal secretion sample and filtered water, the other samples all detected one or two of gene1, gene2, gene3 and gene4 genes, which proves that the corresponding sample sources contain the toxigenic streptococcus. The final criterion for judging that the toxigenic streptococcus suis is positive is that the Ct value obtained by rpoB gene detection is less than 35 cycles, and the Ct value obtained by detecting one or more of gene1, gene2, gene3 and gene4 is less than 35 cycles. If the Ct value obtained by the detection of the rpoB gene is more than 35 cycles, or the Ct values obtained by the detection of the genes gene1, gene2, gene3 and gene4 are more than 35 cycles, the streptococcus suis producing virus is judged to be negative and not detected.
Watch 10
TABLE 11
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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accaatcacc ccaacgtcat 20
<210>5
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tcgaagaagc aggctacaag g 21
<210>6
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
tcaaccaaac ctgcttcaac tg 22
<210>7
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
ccaaaccctc aaagcacagg 20
<210>8
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
tgggagcaat gtgggagatg 20
<210>9
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
acaaaacaca tcgcccttgc 20
<210>10
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
tctctggcaa cccgttcttc 20
Claims (7)
1. The fluorescent quantitative PCR method for detecting the toxigenic streptococcus suis is characterized by comprising the following steps of:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting a streptococcus suis specific gene rpoB, and producing toxin specific genes gene1, gene2, gene3 and gene 4;
s4, reading the Ct value of the amplification; when the Ct value of at least 1 gene in the streptococcus suis specific gene and the toxigenic specific gene is less than 35, the detection result of the toxigenic streptococcus suis is positive; when the Ct value of the streptococcus suis specific gene rpoB is more than 35 and/or the Ct values of the toxigenic specific genes gene1, gene2, gene3 and gene4 are more than 35, the detection result of the toxigenic streptococcus suis is negative.
2. The fluorescent quantitative PCR method for detecting Streptococcus suis according to claim 1, wherein the primers for detecting Streptococcus suis specific gene rpoB are represented by SEQ ID NO 1-2.
3. The fluorescent quantitative PCR method for detecting the toxigenic Streptococcus suis according to claim 1, wherein the detection primer of the toxigenic specific gene1 is shown as SEQ ID NO. 3-4.
4. The fluorescent quantitative PCR method for detecting the toxigenic Streptococcus suis according to claim 1, wherein the detection primer of the toxigenic specific gene2 is shown as SEQ ID NO. 5-6.
5. The fluorescent quantitative PCR method for detecting the toxigenic Streptococcus suis according to claim 1, wherein the detection primer of the toxigenic specific gene3 is shown as SEQ ID NO. 7-8.
6. The fluorescent quantitative PCR method for detecting the toxigenic Streptococcus suis according to claim 1, wherein the detection primer of the toxigenic specific gene4 is shown as SEQ ID NO. 9-10.
7. A fluorescent quantitative PCR kit for detecting the toxigenic streptococcus suis is characterized by comprising a detection primer of the streptococcus suis specific gene rpoB and detection primers of the toxigenic specific genes gene1, gene2, gene3 and gene 4.
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| CN113151516A (en) * | 2021-04-01 | 2021-07-23 | 武汉轻工大学 | Human high-risk zoonosis type streptococcus suis specific sequence, detection primer and application |
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| CN113151516A (en) * | 2021-04-01 | 2021-07-23 | 武汉轻工大学 | Human high-risk zoonosis type streptococcus suis specific sequence, detection primer and application |
| CN113151516B (en) * | 2021-04-01 | 2023-11-28 | 武汉轻工大学 | Human high-risk zoonotic streptococcus suis specific sequence, detection primer and application |
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Application publication date: 20200814 |