CN111534613A - Fluorescent quantitative PCR method for detecting toxigenic mycoplasma pneumoniae and corresponding kit - Google Patents
Fluorescent quantitative PCR method for detecting toxigenic mycoplasma pneumoniae and corresponding kit Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR method for detecting mycoplasma pneumoniae and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the mycoplasma pneumoniae from other mycoplasma genera as well as toxigenic and non-toxigenic mycoplasma pneumoniae genera, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the toxigenic mycoplasma pneumoniae 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 mycoplasma pneumoniae by using specific genes and a corresponding detection kit.
Background
Mycoplasma pneumoniae (Mycoplasma pneumoniae) is a pathogen of human Mycoplasma pneumonia, the incidence rate of the Mycoplasma pneumoniae is highest in teenagers, symptoms such as muscular soreness, anorexia, pharyngalgia, headache, fever, fatigue and the like exist in the early stage of the pneumonia, and the Mycoplasma pneumoniae can occur all the year round but is mostly in autumn and winter.
The mycoplasma pneumoniae is a mycoplasma genus, is the smallest pathogenic microorganism known to live independently between bacteria and viruses, has no cell wall, is in various forms such as a sphere, a mycoplasma and a thread, and can survive for only a few hours at 37 ℃.
The pathogenic mechanism of mycoplasma pneumoniae firstly attaches to the surface of host cells, damages cell membranes of the host cells by microtubules, releases metabolites to dissolve and die epidermal cells, and further causes mycoplasma pneumonia, and the clinical symptoms of the mycoplasma pneumoniae on the respiratory system include mild nasal obstruction and nasal discharge of the nose and moderate congestion of the pharynx. Eardrum is usually congested, and approximately 15% of eardrum is afflicted with myringitis. The cervical lymph nodes may swell. Meanwhile, mycoplasma pneumonia can be associated with multi-system, multi-organ damage, such as: (1) skin lesions may manifest as maculopapules, water sores, etc.; (2) gastrointestinal system symptoms such as emesis, diarrhea and liver function impairment; (3) hemolytic anemia of the blood system; (4) multiple radiculitis, meningoencephalitis, cerebellar injury and the like occur in the central nervous system; (5) the cardiovascular system has myocarditis and pericarditis.
The chest X-ray examination of patients with mycoplasma pneumonia varies greatly, the clinical manifestations may be slight, and the white blood cell amount is different in the routine examination of blood. Detection of pathogens is required for a definitive diagnosis. At present, the diagnosis of mycoplasma pneumonia in China mainly depends on serological detection. In recent years, the mature technology of new functional gene chips brings about the application of a great deal of microbial detection in environment or medical clinic, and the project is the development of gene chips produced in China by the basic exclusive chip technology and aims at the efficient and high-precision detection of mycoplasma pneumoniae.
Disclosure of Invention
One of the objects of the present invention is to provide a fluorescent quantitative PCR method for detecting mycoplasma pneumoniae.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting mycoplasma pneumoniae specific genes rpoB and gene4 and toxin-producing specific genes gene1, gene2 and gene 3;
s4, reading the Ct value of the amplification; when Ct values of at least 1 gene in the mycoplasma pneumoniae specificity genes and at least 1 gene in the virus-producing specificity genes are both less than 35, the detection result of the virus-producing mycoplasma pneumoniae is positive; and when the Ct value of the Mycoplasma pneumoniae specific genes rpoB and gene4 is more than 35 and/or the Ct value of the toxigenic specific genes gene1, gene2 and gene3 is more than 35, the detection result of the Mycoplasma pneumoniae is negative.
As a preferred embodiment, the Mycoplasma pneumoniae specific gene rpoB
The detection primer of (NCBI Accession Number: MG995115.1) is shown in SEQ ID NO: 1-2:
SEQ ID NO:1(5'-TGCGGTTGGTGAATTGCTTG-3')；
SEQ ID NO:2(5'-GAAAGCGGGTTGTGTTGGTC-3')。
the detection primer of the mycoplasma pneumoniae specific gene4(NCBI access Number: VEU57495.1) is shown as SEQ ID NO. 3-4: SEQ ID NO 3
(5'-AGAAAACGTGGGCAGGGAAC-3')；
SEQ ID NO:4(5'-ACTGGACTCTGCAAAGCGAC-3')。
The detection primer of the mycoplasma pneumoniae specific gene1(NCBI access Number: VEU56921.1) is shown as SEQ ID NO: 5-6: SEQ ID NO 5
(5'-AGTTTTGCAATCATTGGTGGCT-3')；
SEQ ID NO:6(5'-GATAATGTAGCTGCTCCAAGGC-3')。
The detection primer of the mycoplasma pneumoniae specific gene2(NCBI access Number: VEU56963.1) is shown as SEQ ID NO: 7-8:
SEQ ID NO:7(5'-CAAATCCCGAGCTTTCAAGGC-3')；
SEQ ID NO:8(5'-ACAAGCCAGTTACAACACCTACT-3')。
the detection primer of the toxin-producing specific gene3 is shown in SEQ ID NO. 9-10:
SEQ ID NO:9(5'-GCCCTAGCCCAAGATACAAGT-3')(NCBI Accession Number：VEU57467.1)；
SEQ ID NO:10(5'-GGCTTTCCCTCTGCAACAAG-3')(NCBI Accession Number：VEU57468.1)。
another objective of the invention is to provide a fluorescent quantitative PCR kit for detecting mycoplasma pneumoniae producing viruses, which comprises the detection primers of the mycoplasma pneumoniae specific genes rpoB and gene4 and the detection primers of the viruses producing specific genes gene1, gene2 and gene 3.
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 Mycoplasma pneumoniae and other Mycoplasma genera, toxigenic and non-toxigenic Mycoplasma genera, specific mycoplasma genes capable of distinguishing Mycoplasma pneumoniae and other Mycoplasma genera, toxigenic and non-toxigenic Mycoplasma genera are excavated, whether toxigenic Mycoplasma pneumoniae exists in a fungus sample and an environment sample or not is accurately judged through detecting the specific genes, the early large data mining and the comparison between different species are provided, and the selected specific genes have the specificity of species and genus.
(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 two-part detection strategy can judge mycoplasma pneumoniae and other mycoplasma, toxigenic and non-toxigenic mycoplasma step by step, two positive detections are needed for judging the mycoplasma pneumoniae is finally determined as toxigenic mycoplasma, 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 source samples in various fields of environmental water body samples, sludge samples, food spot checks and the like, accurately judges whether the samples contain the mycoplasma pneumoniae, and is convenient, rapid and sensitive to operate. The detection kit can be applied to clinical, domestic water monitoring, food safety and other aspects of rapid detection, 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, mycoplasma pneumoniae can be correctly distinguished from other mycoplasma species, toxigenic and non-toxigenic mycoplasma species, thereby accurately detecting and judging toxigenic mycoplasma pneumoniae.
In some embodiments, by extracting and detecting 10, 100, 1000 and 10000 positive bacteria, mycoplasma pneumoniae and other mycoplasma, toxigenic and non-toxigenic mycoplasma can be accurately distinguished, thereby accurately detecting and judging toxigenic mycoplasma pneumoniae. 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, samples of serum, skin secretion, vaginal secretion, urine and filtered water are collected, and the specific genes can be detected to quickly and accurately detect whether the samples contain the mycoplasma pneumoniae for toxigenic pneumonia, so that the method is convenient and quick.
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 fluorescent quantitative PCR method and the kit for detecting the mycoplasma pneumoniae can be widely applied to a plurality of inspection and quarantine categories such as clinical monitoring, drinking water monitoring, food safety monitoring and the like, and can provide a convenient, quick, accurate and efficient monitoring product based on gene detection for daily life.
Example 1 Standard Curve for detection primer amplification
1. Cultivation of microorganisms
The peptone water with alkali was used as the culture medium of mycoplasma pneumoniae, and the peptone water with pH of 8.8-9.0 or the plate had 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
The DNA extracted from Mycoplasma pneumoniae is used for amplification by primers of rpoB gene, gene1 gene, gene2 gene, gene3 gene and gene4 gene respectively, the amplification products are treated by adding A, T4 ligase is used for connecting into a T vector, and after competent cells are transduced, the plasmid is amplified in 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 coating and amplifying standard clone bacteria of primer amplification products corresponding to rpoB gene, gene1 gene, gene2 gene, gene3 gene and gene4 gene, collecting monoclonal bacterial plaques, culturing the bacterial plaques by LB culture and amplifying overnight to finally 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 gene, gene2 gene, gene3 gene and gene4 gene are optimized primer design and a reaction system. Linear standard curve confidence R2>0.990 percent, the high amplification efficiency is 95-107 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
The qPCR amplification result of 5 pairs of primers shows that Mycoplasma pneumoniae shows positive results of rpoB gene and gene4 gene, and other Mycoplasma or Mycoplasma all show negative results. Therefore, the detection of rpoB gene and gene4 gene was performed for Mycoplasma pneumoniae and other Mycoplasma, and it was possible to distinguish Mycoplasma pneumoniae from other Mycoplasma. Meanwhile, the detection results of the gene1 gene, the gene2 gene and the gene3 gene show that the toxigenic mycoplasma pneumoniae shows positive detection results, and other non-toxigenic mycoplasma pneumoniae and other mycoplasma show negative detection results. Therefore, the detection of the gene1, gene2 and gene3 genes can better distinguish the toxigenic and non-toxigenic mycoplasma genera. In conclusion, primers corresponding to rpoB gene and gene4 gene can satisfy the judgment of detection of Mycoplasma pneumoniae among Mycoplasma, and primers corresponding to gene1 gene, gene2 gene and gene3 gene can satisfy the judgment of detection of toxigenic and non-toxigenic Mycoplasma.
TABLE 6
TABLE 7
EXAMPLE 3 sensitivity of the detection System
1. Cultivation of microorganisms
The peptone water with alkali was used as the culture medium of mycoplasma pneumoniae, and the peptone water with pH of 8.8-9.0 or the plate had 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
The results of qPCR amplification with 5 pairs of primers showed that mycoplasma pneumoniae exhibited positive results for one or both of rpoB gene and gene4 gene in the range from 10 bacteria to 10000 bacteria. Therefore, detection of rpoB gene and gene4 gene can achieve detection of Mycoplasma pneumoniae in a range of preferably 10 bacteria to 10000 bacteria. Meanwhile, in the range from 10 bacteria to 10000 bacteria, the detection results of the gene1, the gene2 and the gene3 show that the toxigenic Mycoplasma pneumoniae shows positive detection results, and other non-toxigenic Mycoplasma pneumoniae shows negative detection results. Therefore, the detection of the gene1, gene2 and gene3 can better achieve the detection and the differentiation of toxigenic and non-toxigenic mycoplasma in the range from 10 bacteria to 10000 bacteria. In summary, the detection of the mycoplasma pneumoniae producing virus can be well completed through the matching detection of the rpoB gene, the gene4 gene, the gene1 gene, the gene2 gene and the gene3 gene, the detection range can be from 10 strains to 10000 strains, the higher concentration of the strains can be diluted to the optimal detection range through a dilution method, and the expected detection requirements can be met.
TABLE 8
TABLE 9
Example 4 detection of environmental samples
1. Clinical sample Collection and extraction of genomic DNA
Different serum, skin secretion, vaginal secretion, urine and filtered water were collected separately and genomic DNA extraction was performed according to QIAamp DNA Mini Kit instructions from Qiagen.
qPCR detection of 2.5 target genes
The same as in example 2.
3. Analysis of results
The qPCR amplification result of 5 pairs of primers shows that except filtered water, other samples detect one or two of rpoB gene and gene4 gene, and the corresponding sample sources are proved to contain the mycoplasma pneumoniae. Except for filtered water, serum 3, serum 4, skin secretion 1, vaginal secretion and urine, one or more genes of gene1, gene2 and gene3 were detected in the samples, which proves that the corresponding sample sources contain toxigenic mycoplasma pneumoniae. The final criterion for judging the mycoplasma pneumoniae is that the Ct value obtained by detecting one or two of rpoB gene and gene4 is less than 35 cycles, and the Ct value obtained by detecting one or more of gene1 gene, gene2 gene and gene3 gene is less than 35 cycles. If the Ct value obtained by detecting the rpoB gene and the gene4 gene is more than 35 cycles, or the Ct value obtained by detecting the gene1 gene, the gene2 gene and the gene3 gene is more than 35 cycles, the mycoplasma pneumoniae 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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Claims (7)
1. A fluorescent quantitative PCR method for detecting mycoplasma pneumoniae is characterized by comprising the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting Mycoplasma pneumoniae specific genes rpoB and gene4, and toxin-producing specific genes gene1, gene2 and gene 3;
s4, reading the Ct value of the amplification; when Ct values of at least 1 gene in the mycoplasma pneumoniae specificity genes and at least 1 gene in the virus-producing specificity genes are both less than 35, the detection result of the virus-producing mycoplasma pneumoniae is positive; and when the Ct value of the Mycoplasma pneumoniae specific genes rpoB and gene4 is more than 35 and/or the Ct value of the toxigenic specific genes gene1, gene2 and gene3 is more than 35, the detection result of the Mycoplasma pneumoniae is negative.
2. The fluorescent quantitative PCR method for detecting mycoplasma pneumoniae as claimed in claim 1, wherein the detection primer of mycoplasma pneumoniae specific gene rpoB is shown in SEQ ID NO 1-2.
3. The fluorescent quantitative PCR method for detecting mycoplasma pneumoniae as claimed in claim 1, wherein the detection primer of the mycoplasma pneumoniae specific gene4 is shown as SEQ ID NO. 3-4.
4. The fluorescent quantitative PCR method for detecting mycoplasma pneumoniae as claimed in claim 1, wherein detection primers of the toxigenic specific gene1 are shown in SEQ ID NO 5-6.
5. The fluorescent quantitative PCR method for detecting mycoplasma pneumoniae as claimed in claim 1, wherein detection primers of the toxigenic specific gene2 are shown in SEQ ID NO 7-8.
6. The fluorescent quantitative PCR method for detecting the toxigenic Mycoplasma pneumoniae of claim 1, wherein a detection primer of the toxigenic specific gene3 is shown as SEQ ID NO. 9-10.
7. A fluorescent quantitative PCR kit for detecting mycoplasma pneumoniae producing viruses is characterized by comprising detection primers of mycoplasma pneumoniae specific genes rpoB and gene4 and detection primers of virus producing specific genes gene1, gene2 and gene 3.
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| CN110300578A (en) * | 2016-12-23 | 2019-10-01 | 英特维特国际股份有限公司 | For treating the compound of ox or porcine respiratory disease |
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Non-Patent Citations (3)
| Title |
|---|
| PÁLMA SILLÓ等: "Eosinophilic Fasciitis associated with Mycoplasma arginini infection", 《J CLIN MICROBIOL.》 * |
| 刘茂军等: "猪肺炎支原体不同毒力株黏附因子基因序列分析", 《江苏农业学报》 * |
| 杨发龙等: "绵羊肺炎支原体毒力相关基因预测与分析", 《黑龙江畜牧兽医》 * |
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Application publication date: 20200814 |