CN111518926A - Fluorescent quantitative PCR method for detecting toxigenic mycoplasma hominis and corresponding kit - Google Patents
Fluorescent quantitative PCR method for detecting toxigenic mycoplasma hominis and corresponding kit Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR method for detecting toxigenic mycoplasma hominis and a corresponding kit. The invention skillfully uses specific gene detection to distinguish the human mycoplasma from other mycoplasma genera as well as toxigenic and non-toxigenic mycoplasma genera, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the toxigenic mycoplasma 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 toxigenic mycoplasma and a corresponding detection kit.
Background
Mycoplasma hominis (Mycoplasma hominis), a species of Mycoplasma, is present in the urinary system and genitals and can cause urinary infections and genital inflammation.
Mycoplasma hominis has no cell wall and precursor, and has a very small genome in its cells. The mycoplasma is propagated by two divisions, the shape is various, the bacterial colony is large, the diameter is 300-1000 microns, and the typical bacterial colony has the appearance of a fried egg. Culturing with culture medium. The nutritional requirements are higher than for bacteria. Because it has no cell wall, it is insensitive to antibiotics that affect cell wall synthesis, such as penicillin and the like. Mycoplasma is also poorly heat resistant and is normally inactivated by treatment at 55 ℃ for 15 minutes.
The infection caused by mycoplasma in human body has urethral stabbing pain, urinary urgency and urinary frequency with different degrees in clinical manifestation, which causes urinary system infection. The male often has prostate infection, and perineum distending pain appears. Female patients often have genital inflammation that spreads around the cervix. If people find mycoplasma positive during examination, but if no accompanying symptoms exist, people do not need to be alarmed excessively, and the mycoplasma positive is in a normal carrying state and does not mean to treat diseases.
In the related detection aspect, nucleic acid amplification (PCR) is mainly carried out based on MgPa adhesin gene or 16S rRNA gene, and in recent years, the mature technology of new functional gene chips brings about that a great deal of microbial detection is applied to environment or medical clinic, while the project is that the basic exclusive chip technology develops national gene chips to carry out high-efficiency and high-precision detection on human mycoplasma.
Disclosure of Invention
One of the objects of the present invention is to provide a fluorescent quantitative PCR method for detecting a toxigenic Mycoplasma hominis.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting specific genes rpoB, gene1, gene2 and gene3 of the human mycoplasma, and detecting a specific gene4 of the toxigenicity;
s4, reading the Ct value of the amplification; when Ct values of at least 1 gene in the mycoplasma hominis specific genes and the toxigenic specific gene are both smaller than 35, the detection result of the toxigenic mycoplasma hominis is positive; when the Ct value of the mycoplasma hominis specific genes rpoB, gene1, gene2 and gene3 is more than 35 and/or the Ct value of the toxigenic specific gene4 is more than 35, the detection result of the mycoplasma hominis is negative.
As a preferred technical scheme, a detection primer of the specific gene rpoB (NCBI accession number: AF443616.3) of the human mycoplasma is shown as SEQ ID NO 1-2;
SEQ ID NO:1(5'-ACATTCAAAGGCTGGCGAAG-3')；
SEQ ID NO:2(5'-CACCTGCTGTTACTTCTGAACC-3')。
the detection primer of the human mycoplasma specific gene1(NCBI Accession Number: AUW36898.1) is shown as SEQ ID NO. 3-4;
SEQ ID NO:3(5'-CATGCCCATAAGTGCCTTACC-3')；
SEQ ID NO:4(5'-ATAGTACGCAGCAAACCCGG-3')。
the detection primer of the human mycoplasma specific gene2(NCBI Accession Number: AYN65194.1) is shown as SEQ ID NO. 5-6;
SEQ ID NO:5(5'-TGACTACATTACACCAGCTCGT-3')；
SEQ ID NO:6(5'-ACGTTGATTCCTCTGTCCTTCAT-3')。
the detection primer of the specific gene3(NCBI Accession Number: AYN65700.1) of the human mycoplasma is shown as SEQ ID NO: 7-8;
SEQ ID NO:7(5'-AGATGCACGCTTACCTCTTACT-3')；
SEQ ID NO:8(5'-AGATCTGCTTTGTCGCTTTTGT-3')。
the detection primer of the toxin-producing specific gene4 is shown as SEQ ID NO. 9-10;
SEQ ID NO:9(5'-TGCTTGCTAAACCTAATGCCAC-3')(NCBI Accession Number：AYN65533.1)；
SEQ ID NO:10(5'-CCGCCTGATAAATTTGCACCC-3')(NCBI Accession Number：AYN65534.1)。
another objective of the invention is to provide a fluorescent quantitative PCR kit for detecting toxigenic Mycoplasma hominus, which comprises the detection primers of the specific genes rpoB, gene1, gene2 and gene3 of Mycoplasma hominus and the detection primer of the specific gene4 of toxigenic Mycoplasma hominus.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, through a large amount of early-stage research data on microorganisms of human mycoplasma and other mycoplasma, toxigenic mycoplasma and non-toxigenic mycoplasma, orphan genes capable of distinguishing the specificity of the human mycoplasma from the specificity of the other mycoplasma, toxigenic mycoplasma and non-toxigenic mycoplasma are excavated, and whether a clinical sample contains toxigenic human mycoplasma is accurately judged through detecting the specific genes, so that the early-stage large data mining and 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 of the invention can judge the mycoplasma hominis and other mycoplasma, toxigenic and non-toxigenic mycoplasma step by step, and the mycoplasma hominis is finally judged to be toxigenic only by judging two positive detections at the same time, so that 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 in-vitro secretion, excrement and the like in clinical medicine, accurately judges whether the clinical samples contain the toxigenic mycoplasma hominis, 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, one can correctly distinguish between mycoplasma hominis and other mycoplasma species, toxigenic and non-toxigenic mycoplasma species, thereby accurately detecting and determining toxigenic mycoplasma hominis.
In some embodiments, by extracting and detecting 10, 100, 1000, and 10000 positive bacteria, mycoplasma hominis can be accurately distinguished from other mycoplasma species, toxigenic and non-toxigenic mycoplasma species, thereby accurately detecting and determining toxigenic mycoplasma hominis. 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, clinical samples including vaginal secretion, skin secretion, urine, feces and filtered water are collected, and specific genes are detected, so that whether the samples contain the toxigenic mycoplasma hominis can be quickly and accurately detected, and 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 method and the kit for detecting the fluorogenic quantitative PCR of the toxigenic mycoplasma can be widely applied to a plurality of inspection and quarantine categories such as in-vivo and in-vitro secretion detection, excrement detection, drinking water monitoring and the like of a human body clinically, and can provide a convenient, quick, accurate and efficient gene detection-based monitoring product for clinical medicine and daily lives.
Example 1 Standard Curve for detection primer amplification
1. Cultivation of microorganisms
The alkaline peptone water is used as a culture medium of the human mycoplasma, and the alkaline peptone water with the pH value of 8.8-9.0 or a flat plate 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 Mycoplasma hominis is used and amplified with primers for rpoB gene, gene1, gene2, gene3 and gene4, respectively, the amplification product is treated with A, T4 ligase is used to ligate into T vector, and after competent cells are transduced, the plasmid is 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 clone bacteria of amplification products of primers corresponding to rpoB gene, gene1, gene2, gene3 and gene4, collecting 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 instructions 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
| Name of gene | Slope of | Intercept of a beam | Efficiency of amplification | Confidence level R2 |
| rpoB | -3.2952 | 35.857 | 101.13％ | 0.9981 |
| gene1 | -3.2542 | 36.092 | 102.91％ | 0.9976 |
| gene2 | -3.2841 | 35.019 | 101.60％ | 0.9976 |
| gene3 | -3.4243 | 35.996 | 95.90％ | 0.9985 |
| gene4 | -3.4647 | 35.556 | 94.37％ | 0.9988 |
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 table 6 and table 7, the qPCR amplification results with 5 pairs of primers showed that the human mycoplasma genus exhibited positive results for one to many of rpoB gene, gene1, gene2, and gene3, while the other mycoplasma genera all showed negative results. Therefore, the detection of rpoB genes, gene1, gene2 and gene3 was performed for the human mycoplasma and other mycoplasma, and the human mycoplasma and other mycoplasma can be distinguished well. Meanwhile, as a result of the detection by gene4, toxigenic human mycoplasma species showed positive detection results, while other non-toxigenic human mycoplasma species and other mycoplasma species showed negative results. Therefore, the detection of gene4 can be performed to better distinguish toxigenic and non-toxigenic mycoplasma. In conclusion, the primers corresponding to rpoB gene, gene1, gene2 and gene3 satisfy the judgment of detection of human Mycoplasma among Mycoplasma, and the primer corresponding to gene4 satisfies 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 alkaline peptone water is used as a culture medium of the human mycoplasma, and the alkaline peptone water with the pH value of 8.8-9.0 or a flat plate 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 below, the qPCR amplification results with 5 pairs of primers showed that the human mycoplasma genus exhibited positive results for one to more genes of rpoB gene, gene1, gene2, and gene3 in the range of 10 bacteria to 10000 bacteria. Therefore, detection of rpoB gene, gene1, gene2 and gene3 enables detection of a human mycoplasma genus in a range of 10 bacteria to 10000 bacteria. Meanwhile, in the range from 10 bacteria to 10000 bacteria, the detection result of gene4 shows that the mycoplasma producing virus shows positive detection result, and other mycoplasma not producing virus shows negative detection result. Therefore, the detection of gene4 can be performed to achieve detection and differentiation of mycoplasma species that are toxigenic and non-toxigenic in a range from 10 bacteria to 10000 bacteria. In summary, the detection of toxigenic mycoplasma hominis 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, higher strain concentration can be diluted to an optimal detection range through a dilution method, and 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 vaginal secretions, skin secretions, urine, feces and filtered water were collected separately and genomic DNA extraction was performed as instructed by 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 results of the qPCR amplification with 5 pairs of primers showed that, in addition to filtered water and one stool sample, 1 or more of rpoB gene, gene1, gene2 and gene3 were detected from all samples, demonstrating that the corresponding sample sources contained mycoplasma hominis. The gene4 gene was detected in all samples except two vaginal secretion samples, two skin secretion samples, one urine sample, one feces sample and the filtered water sample, which proved that the corresponding sample source contained toxigenic mycoplasma hominis. The final criterion for judging the mycoplasma toxigenic is that the Ct value obtained by detecting 1 or more of rpoB gene, gene1, gene2 and gene3 is less than 35 cycles, and the Ct value obtained by detecting gene4 is less than 35 cycles. If the Ct value detected by the rpoB gene, the gene1, the gene2 and the gene3 is more than 35 cycles, or the Ct value detected by the gene4 is more than 35 cycles, the mycoplasma toxigenic hominis 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 toxigenic mycoplasma hominis is characterized by comprising the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting specific genes rpoB, gene1, gene2 and gene3 of the human mycoplasma, and detecting a specific gene4 of the toxigenicity;
s4, reading the Ct value of the amplification; when Ct values of at least 1 gene in the mycoplasma hominis specific genes and the toxigenic specific gene are both smaller than 35, the detection result of the toxigenic mycoplasma hominis is positive; when the Ct value of the mycoplasma hominis specific genes rpoB, gene1, gene2 and gene3 is more than 35 and/or the Ct value of the toxigenic specific gene4 is more than 35, the detection result of the mycoplasma hominis is negative.
2. The fluorescent quantitative PCR method for detecting toxigenic Mycoplasma hominis according to claim 1, wherein the detection primer of the Mycoplasma hominus specific gene rpoB is shown as SEQ ID NO 1-2.
3. The fluorescent quantitative PCR method for detecting toxigenic Mycoplasma hominis according to claim 1, wherein the detection primer of the Mycoplasma hominus specific gene1 is shown as SEQ ID NO. 3-4.
4. The fluorescent quantitative PCR method for detecting toxigenic Mycoplasma hominis according to claim 1, wherein the detection primer of the Mycoplasma hominus specific gene2 is shown as SEQ ID NO. 5-6.
5. The fluorescent quantitative PCR method for detecting toxigenic Mycoplasma hominis according to claim 1, wherein the detection primer of the Mycoplasma hominus specific gene3 is shown as SEQ ID NO. 7-8.
6. The fluorescent quantitative PCR method for detecting the toxigenic mycoplasma hominis according to claim 1, wherein a detection primer of the toxigenic specific gene4 is shown as SEQ ID NO. 9-10.
7. A fluorescent quantitative PCR kit for detecting toxigenic mycoplasma hominis is characterized by comprising detection primers of the mycoplasma hominis specific genes rpoB, gene1, gene2 and gene3 and detection primers of the toxigenic specific gene 4.
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