CN111518925A

CN111518925A - Fluorescent quantitative PCR method for detecting toxigenic tetanus bacillus and corresponding kit

Info

Publication number: CN111518925A
Application number: CN201911100944.5A
Authority: CN
Inventors: 曹亮; 张智闵; 连政汉; 蒋华; 束文圣
Original assignee: Guangdong Magigene Technology Co ltd
Current assignee: Guangdong Magigene Technology Co ltd
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2020-08-11

Abstract

The invention discloses a fluorescence quantitative PCR method for detecting toxigenic tetanus bacillus and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the tetanus bacillus from other genera as well as toxigenic and non-toxigenic tetanus bacillus, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting toxigenic tetanus bacillus 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

Fluorescent quantitative PCR method for detecting toxigenic tetanus bacillus and corresponding kit

Technical Field

The invention belongs to the technical field of molecular detection, and particularly relates to a method for carrying out fluorescence quantitative PCR detection on toxigenic tetanus bacillus through specific genes and a corresponding detection kit.

Background

Tetanus bacillus (clostridium tetani) is a pathogenic bacterium causing tetanus, is abundantly present in human and animal intestines, and causes diseases through wound infection after soil contamination by feces. The latent period of tetanus is indefinite, the short period is 1-2 days, the long period is 2 months, and the average period is 7-14 days. The shorter the latency, the higher the mortality rate. The fatality rate is about 50%, especially for pregnant women, newborns and the elderly.

The whole body flagella and spores of the tetanus bacillus thallus are round, the body length is about 4-8 um, and the body width is about 0.3-0.5 um. The nutrient requirement is not high, and after the culture is carried out on a common agar plate for 24-48 hours, irregular colonies with the diameter of more than 1mm can be formed, the center is compact, the periphery is loose, and the colonies are similar to feather-shaped colonies.

Tetanus can invade human bodies from wounds and germinate and propagate to cause diseases, but clostridium tetani is anaerobic bacteria and cannot grow in common wounds, and the anaerobic environment of the wounds is an important condition for clostridium tetani infection. Narrow and deep wounds (such as puncture wounds) are polluted by mud or foreign matters, or large-area wounds, burns and necrotic tissues are abundant, and an anaerobic environment is easily caused by local tissue ischemia or simultaneous aerobic bacteria or facultative anaerobic bacteria mixed infection, so that the local oxidation-reduction potential is reduced. Is beneficial to the growth of the tetanus bacillus. Clostridium tetani produces a strong exotoxin, tetanus spastic toxin tetanus. The main clinical examination is to take out the injured tissue part and culture biochemical tests. In recent years, molecular biology techniques have been applied to the detection of clostridium tetani gradually due to the advantages of high sensitivity, high specificity, short experimental period, easy operation, etc., so the development of domestic gene chips for the efficient and high-precision detection of clostridium tetani has been the project.

Disclosure of Invention

One of the objects of the present invention is to provide a fluorescent quantitative PCR method for detecting tetanus toxigenic bacteria.

Specifically, the method comprises the following steps:

s1, collecting a sample;

s2, extracting genome DNA;

s3, detecting specific genes rpoB, gene3 and gene4 of the genus tetanus bacillus and specific genes gene1 and gene2 of toxin production;

s4, reading the Ct value of the amplification; when Ct values of at least 1 gene in the specific genes of the tetanus bacillus and at least 1 gene in the specific genes for generating the toxin are both less than 35, the detection result of the tetanus bacillus for generating the toxin is positive; when the Ct value of the tetanus bacillus specific genes rpoB, gene3 and gene4 is more than 35 and/or the Ct value of the toxigenic specific genes gene1 and gene2 is more than 35, the detection result of the toxigenic tetanus bacillus is negative.

As a preferred embodiment, the specific gene rpoB of the genus tetanus

The detection primer of (NCBI access Number: NZ _ QMBF01000006.1) is shown in SEQ ID NO: 1-2:

SEQ ID NO:1(5'-TGGCAGGAAGACACGGAAAT-3')；

SEQ ID NO:2(5'-ATTTCCGGTTCAGTTGCACC-3')。

the detection primer of the specific gene3 of the genus tetanus is shown in SEQ ID NO. 3-4: SEQ ID NO. 3(5'-AGGAATTGTAGAACCTCACTGCC-3') (NCBI Accession Number: QBD 86639.1);

SEQ ID NO:4(5'-ACTCCTGCTCGTTTTGCCAT-3')(NCBI Accession Number：QBD86640.1)。

the detection primer of the specific gene4 of the genus tetanus is shown in SEQ ID NO: 5-6: SEQ ID NO. 5(5'-AACCCCTGCTTTCCCTGTAC-3') (NCBI Access Number: AAO 35365.1);

SEQ ID NO:6(5'-CCCAAGCTTAAACACCTGCC-3')(NCBI Accession Number：AAO35366.1)。

the detection primer of the toxin-producing specific gene1 is shown in SEQ ID NO: 7-8:

SEQ ID NO:7(5'-GAAAAGCTTGGCGGAACAGG-3')(NCBI Accession Number：QBD87392.1)；

SEQ ID NO:8(5'-AGCACTGCCAATGGAGTTCT-3')(NCBI Accession Number：QBD87393.1)。

the detection primer of the specific gene2 for toxin production (NCBI Accession Number: QBD86663.1) is shown as SEQ ID NO: 9-10:

SEQ ID NO:9(5'-GTGCATGGATGTATGGCCCT-3')；

SEQ ID NO:10(5'-AATCAAGTTGCCAAGAAGCTCC-3')。

another object of the present invention is to provide a fluorescent quantitative PCR kit for detecting tetanus bacteria, which comprises the detection primers for the specific genes rpoB, gene3 and gene4 of the genus tetanus bacteria and the detection primers for the specific genes gene1 and gene2 of the genus tetanus bacteria.

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 the genus tetanus, the genus other than the genus tetanus, the genus toxigenic and the genus non-toxigenic bacteria, specific rod genes capable of distinguishing the genus tetanus from the genus other than the genus tetanus, the genus toxigenic and the genus non-toxigenic bacteria are excavated, whether the bacteria sample and the environment sample contain the toxigenic tetanus is accurately judged through detecting the specific genes, the early large data is mined and the comparison basis among different species is provided, and the selected specific genes have the specificity of the species and the 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 detection method provided by the invention has the advantages that the two-part detection strategy can judge the tetanus bacillus and other bacteria, toxigenic tetanus bacillus and non-toxigenic tetanus bacillus step by step, two items of positive detection are required to be carried out simultaneously for judging the tetanus bacillus as toxigenic tetanus bacillus, 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 such as clinical samples, environmental water samples, food spot inspection and the like, accurately judges whether the samples contain toxigenic tetanus bacillus, and is convenient, rapid and sensitive to operate. The detection kit can be applied to medical and health monitoring, 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, tetanus bacteria can be correctly distinguished from other genera, toxigenic and non-toxigenic, thereby accurately detecting and determining toxigenic tetanus bacteria.

In some embodiments, by extracting and detecting 10, 100, 1000 and 10000 positive bacteria, tetanus bacillus and other genera, toxigenic and non-toxigenic bacillus can be accurately distinguished, thereby accurately detecting and judging toxigenic tetanus bacillus. 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 method is convenient and quick to collect a sample of the blood nourishing serum, the skin secretion, the sputum, the iron rust leaching liquor and the filtered water, and can quickly and accurately detect whether the sample contains the toxin-producing tetanus bacillus through the detection of the specific gene.

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 tetanus bacillus can be widely applied to a plurality of inspection and quarantine categories such as medical and health monitoring, water body 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 lives.

Example 1 Standard Curve for detection primer amplification

1. Cultivation of microorganisms

The alkali peptone water is used as a culture medium of the tetanus bacillus, and the alkali peptone water or the 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 tetanus bacillus was used, amplification was performed with primers for rpoB gene, gene1 gene, gene2 gene, gene3 gene and gene4 gene, respectively, the amplification products were subjected to A addition treatment, T4 ligase was used for ligation into T vector, and after transduction of competent cells, plasmid was 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 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 94-109 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	-3.211	33.319	104.85％	0.9915
gene1	-3.1938	37.022	105.64％	0.9942
					gene2	-3.1243	34.451	108.96％	0.9977
gene3	-3.4584	34.903	94.60％	0.9965
					gene4	-3.1628	34.692	107.10％	0.9996

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 tetanus bacillus shows positive results of rpoB gene, gene3 gene and gene4 gene, and other bacillus shows negative results. Therefore, the detection of rpoB gene, gene3 gene and gene4 gene can be performed for tetanus bacillus and other genera, and thus tetanus bacillus and other genera can be well distinguished. Meanwhile, the detection results of the gene1 gene and the gene2 gene show that the toxigenic tetanus bacillus shows positive detection results, and other non-toxigenic tetanus bacillus and other bacillus show negative detection results. Therefore, the detection of gene1 and gene2 can better distinguish between toxigenic and non-toxigenic bacilli. In conclusion, the primers corresponding to rpoB gene, gene3 and gene4 can satisfy the detection judgment of tetanus bacillus in bacillus, and the primers corresponding to gene1 and gene2 can satisfy the detection judgment of toxigenic and non-toxigenic bacillus.

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 tetanus bacillus, and the alkali peptone water or the 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

The qPCR amplification results of 5 pairs of primers showed that tetanus showed positive results for one or more of rpoB gene, gene3 gene and gene4 gene in the range from 10 bacteria to 10000 bacteria. Therefore, detection of rpoB gene, gene3 and gene4 gene can achieve detection of tetanus bacillus well within the range of 10 bacteria to 10000 bacteria. Meanwhile, in the range from 10 bacteria to 10000 bacteria, the detection results of the gene1 gene and the gene2 gene show that the toxigenic tetanus bacillus shows positive detection results, and other non-toxigenic tetanus bacillus shows negative detection results. Therefore, the detection of the gene1 and the gene2 can better achieve the detection and the differentiation of toxigenic and non-toxigenic tetanus bacillus in the range from 10 bacteria to 10000 bacteria. In summary, the detection of the toxigenic tetanus bacillus can be well completed through the matching detection of rpoB gene, gene1 gene, gene2 gene, gene3 gene and gene4 gene, the detection range can be from 10 strains to 10000 strains, the higher strain concentration 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, sputum, iron rust leach and filtered water were collected separately and genomic DNA was extracted as indicated in 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 more of rpoB gene, gene3 gene and gene4 gene, and the corresponding sample sources are proved to contain tetanus bacillus. Except for serum 3, serum 4, skin secretion 1, sputum and iron rust leaching liquor, one or two of gene1 and gene2 genes are detected in other samples, and the corresponding sample sources are proved to contain toxigenic tetanus bacillus. The final criterion for judging the toxigenic tetanus bacillus to be positive is that the Ct value obtained by detecting one or more of rpoB gene, gene3 and gene4 is less than 35 cycles, and the Ct value obtained by detecting one or two of gene1 and gene2 is less than 35 cycles. If the Ct values obtained by detecting the rpoB gene, the gene3 gene and the gene4 gene are more than 35 cycles, or the Ct values obtained by detecting the gene1 gene and the gene2 gene are more than 35 cycles, the tetanus toxin producing bacillus 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

1. A fluorescence quantitative PCR method for detecting toxigenic tetanus bacillus is characterized by comprising the following steps:

s1, collecting a sample;

s2, extracting genome DNA;

s3, detecting specific genes rpoB, gene3 and gene4 of the genus tetanus bacillus and detecting specific genes gene1 and gene2 of the genus toxigenic bacteria;

2. The fluorescence quantitative PCR method for detecting toxigenic tetanus bacillus according to claim 1, wherein the detection primer of the tetanus bacillus specific gene rpoB is shown as SEQ ID NO 1-2.

3. The fluorescence quantitative PCR method for detecting toxigenic tetanus bacillus according to claim 1, wherein the detection primers of the tetanus bacillus specific gene3 are shown in SEQ ID NO 3-4.

4. The fluorescence quantitative PCR method for detecting toxigenic tetanus bacillus according to claim 1, wherein the detection primers of the tetanus bacillus specific gene4 are shown in SEQ ID NO 5-6.

5. The fluorescence quantitative PCR method for detecting tetanus bacillus as claimed in claim 1, wherein the primer for detecting the specific gene1 is shown in SEQ ID NO 7-8.

6. The fluorescence quantitative PCR method for detecting tetanus bacillus according to claim 1, wherein the primer for detecting the toxin-producing specific gene2 is shown in SEQ ID NO 9-10.

7. A fluorescent quantitative PCR kit for detecting toxigenic tetanus bacillus is characterized by comprising detection primers of the tetanus bacillus specific genes rpoB, gene3 and gene4 and detection primers of the toxigenic specific genes gene1 and gene 2.