CN109022603A - Detect the real-time fluorescence quantitative PCR nucleic acid sequence and kit of Type B clostridium botulinum - Google Patents

Abstract

The present invention provides a real-time fluorescent quantitative PCR nucleic acid sequence and kit for detecting Clostridium botulinum type B, the nucleic acid sequence includes upstream primer B-F, downstream primer B-R and probe B-P, with the primers provided by the present invention The detection accuracy of Clostridium botulinum type B with the probe is 100%. When amplifying the DNA of other 25 intestinal pathogenic bacteria and common bacteria, no specific amplification curve appears, and the specificity is good. . The fluorescent quantitative PCR standard curve constructed according to the recombinant plasmid standard product can confirm that its detection sensitivity for Clostridium botulinum type B is 6.91×10 ² copies/µl. In addition, in the real-time quantitative PCR detection system, when the concentration of the recombinant plasmid containing the B toxin gene is in the range of 10 ² to 10 ⁹ copies/µl, there is a good linear relationship between the Ct value of the amplification reaction and the template concentration. The invention has the characteristics of sensitive response, high specificity, rapidity, convenience, high throughput and the like, and provides an effective detection method for Clostridium botulinum type B screening, large-scale quarantine and epidemiological investigation.

Description

Real-time fluorescent quantitative PCR nucleic acid sequence and kit for detecting Clostridium botulinum type B

technical field

The invention relates to molecular detection of Clostridium botulinum, in particular to a real-time fluorescence quantitative PCR nucleic acid sequence and a kit for detecting Clostridium botulinum type B.

Background technique

Clostridium botulinum ( C. botulinum ) is a Gram-positive, anaerobic, flagellated, acapsulated, spore-shaped, stubby, oval-shaped bacterium. It was first discovered from sausage by Belgian scholar Van Ermengemcong in 1896, and confirmed that it can produce exotoxin-botulinum toxin (Botulinum toxin), causing human and animal poisoning. This bacterium is widely present in the sediments of soil, oceans and lakes, as well as in the intestines, feed and food of mammals, birds and fish. Clostridium botulinum cannot grow in living organisms. When the environment is anaerobic and has proper nutrition, it can grow and reproduce to produce botulinum toxin. When humans and animals eat food and feed containing this toxin, symptoms of poisoning can occur. According to the antigenicity of botulinum toxin, Clostridium botulinum can be divided into 7 types of AG, among which A, B, E, and F are human poisoning types, C and D are animal and poultry poisoning types, and G type is extremely rare. H type has recently been found in infant botulism cases.

Botulinum toxin is the most severe neurotoxin known so far. The food poisoning caused by it has been found in more than a dozen provinces and regions in my country. The fatality rate is extremely high and it endangers food safety. At present, the detection methods for Clostridium botulinum mainly include immunological methods such as isolation and culture, mouse animal experiments, indirect ELISA for toxins, and molecular biological methods such as ordinary PCR, which are time-consuming, labor-intensive and low-sensitivity.

Real-time fluorescent quantitative polymerase chainreaction (qPCR) technology refers to adding a fluorescent group to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve method. Since qPCR is a real-time quantitative detection of pathogenic gene nucleic acid, it has unique advantages over immunological methods such as chemiluminescence, time-resolved, and protein chips. The invention designs primers and probes for the toxin gene of Clostridium botulinum type B, establishes a real-time fluorescent quantitative PCR rapid detection system, and provides a new technical means for the rapid detection of Clostridium botulinum type B.

Contents of the invention

The object of the present invention is to provide a real-time fluorescent quantitative PCR nucleic acid sequence and kit for detecting Clostridium botulinum type B, and provide a new technical means for the rapid detection of Clostridium botulinum type B.

To achieve the above object, the present invention is achieved through the following technical solutions:

Detect the nucleic acid sequence of Clostridium botulinum type B, the nucleic acid sequence includes primers and probes, the primers are the upstream primer B-F and the downstream primer B-R for detecting the B toxin gene of Clostridium botulinum, and the probes are for detecting botulinum The probe B-P of the Clostridium B toxin gene, the upstream primer B-F, the downstream primer B-R and the nucleotide sequences of the probe B-P are respectively shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, specifically:

Upstream primers B-F: 5'- cacaaacattgctagtgtaactgttaataa -3', (SEQ ID No: 1);

Downstream primer B-R: 5'- ctatagtctcattttcatttaaaactggc -3', (SEQ ID No: 2);

Probe B-P: 5'-cagtaatccaggagaagtggagcgaaaaaagg -3', (SEQ ID No: 3); the 5' end of the probe B-P is labeled with a reporter fluorescent group JOE, and the 3' end is labeled with a quencher fluorescent group BHQ2. The length of the target fragment amplified by the above primers is 130bp.

The present invention also provides a real-time fluorescent quantitative PCR kit for detecting Clostridium botulinum type B, comprising the following components:

(1) qPCR reaction system components, including upstream primers B-F shown in sequence SEQ ID No: 1, downstream primers B-R shown in sequence SEQ ID No: 2 and probes B-P shown in sequence SEQ ID No: 3 , where the 5' end of the probe B-P is labeled with the reporter fluorophore JOE, and the 3' end is labeled with the quencher fluorophore BHQ2.

(2) Negative control, using TE buffer, the TE buffer is 10mM Tris-HCl, 1mM EDTA.

(3) Positive control, Clostridium botulinum type B recombinant plasmid standard.

(4) Dilution buffer. TransNGS ^TM Library Dilution Buffer from Quanshijin Biological Company was selected.

Further, the above-mentioned qPCR reaction system components also include reagents and enzymes required for qPCR reaction, preferably, the qPCR reaction system also includes qPCR SuperMix, and the qPCR SuperMix contains dNTPs, Taq DNA polymerase and PCR reaction buffer. qPCR SuperMix was purchased from Quanshijin Biological Company.

The present invention also provides a real-time fluorescent quantitative PCR detection method for detecting Clostridium botulinum type B, comprising the following steps:

(1) Extract the bacterial genomic DNA of the sample to be tested;

Bacterial genomic DNA was extracted using a kit from Promega Technology Co., Ltd., operated according to the instructions, and stored at -20°C for future use. Extraction can also be performed using methods known in the art for extracting DNA.

(2) Perform qPCR amplification on the extracted sample DNA; where the upstream primers B-F shown in the sequence SEQ ID No: 1, the downstream primers B-R shown in the sequence SEQ ID No: 2 and the sequence SEQ ID No: 3 are used As shown in the probe B-P, the 5' end of the probe B-P is labeled with the reporter fluorescent group JOE, and the 3' end is labeled with the quencher fluorescent group BHQ2.

Table 1 qPCR reaction system:

name Final concentration Sample volume (μl) qPCR SuperMix 2× 12.5 Primers B-F 10µM 0.25 Primers B-R 10µM 0.25 Probe B-P 10µM 0.5 template DNA ≥10 ³ copies/µl 0.5 ddH ₂ O 10.5 total 25

The reaction conditions were: pre-denaturation at 95°C for 2min, denaturation at 95°C for 10s, extension at 60°C for 30s, a total of 45 cycles.

(3) Judgment of results:

Result analysis condition setting: the threshold setting principle is that the threshold line just exceeds the highest point of the normal negative control amplification curve. Ct value ≤ 35, and there is an obvious amplification curve is positive, indicating the presence of Clostridium botulinum type B in the sample; no Ct value, and no amplification curve, it is negative. If the Ct value of the sample is >35, it is recommended to redo the test. If there is no Ct value in the redo test, it is negative, otherwise it is positive.

Beneficial effects of the present invention:

1. The primers and probes provided by the present invention can be used for qualitative and quantitative analysis of Clostridium botulinum type B, with sensitive response and good specificity.

2 It can detect a large number of samples at the same time, and the results can be observed within 2 hours. It is fast, sensitive, convenient, and high-throughput. an effective means of detection.

Description of drawings

Figure 1 is a graph of the fluorescence signal of a B toxin gene recombinant plasmid standard product.

Figure 2 is the standard curve corresponding to the logarithmic value of the B toxin gene recombinant plasmid standard product-Ct.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but these examples are only limited to illustrate the present invention and are not intended to limit the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1 Detects the design of special primers and probes for Clostridium botulinum type B

According to the Clostridium botulinum type B toxin gene sequence published on GenBank, use the CLUSTLAW software to perform multiple alignments on the B toxin gene of Clostridium botulinum, select its stable conserved region as the detection target sequence, and design a pair of Primers and a probe, wherein the sequence of primers B-F is shown in SEQ ID No: 1; the sequence of primers B-R is shown in SEQ ID No: 2; the sequence of probes B-P is shown in SEQ ID No: 3. The 5' end of the probe B-P is labeled with the reporter fluorescent group JOE, and the 3' end is labeled with the quencher fluorescent group BHQ2. Finally, blast comparison with GenBank to determine the specificity of primers and probes, and then handed over to Shanghai Sangon Biotechnology Co., Ltd. for synthesis.

Embodiment 2 detects the composition of the kit of the fluorescent quantitative PCR of Clostridium botulinum type B

(1) qPCR reaction system components, including upstream primers B-F shown in sequence SEQ ID No: 1, downstream primers B-R shown in sequence SEQ ID No: 2 and probes B-P shown in sequence SEQ ID No: 3 , wherein the 5' end of the probe B-P is labeled with the reporter fluorescent group JOE, and the 3' end is labeled with the quencher fluorescent group BHQ2; the qPCR reaction system also includes qPCRSuperMix, the qPCR SuperMix contains dNTPs, Taq DNA polymerase and PCR reaction buffer. qPCR SuperMix was purchased from Quanshijin Biological Company.

(2) Negative control, using TE buffer, the composition is as follows: 10mM Tris-HCl, 1mM EDTA

(3) Positive control, Clostridium botulinum type B recombinant plasmid standard.

(4) Dilution buffer. TransNGS ^TM Library Dilution Buffer from Quanshijin Biological Company was selected.

Construction and preparation of embodiment 3 plasmid standard

Using B-F and B-R as primers, carry out PCR amplification, the target product fragment is purified and recovered by cutting gel, and then connected to pMD-18T vector, transformed into JM109 competent cells, screened positive clones, verified by PCR and sequenced for final confirmation.

Example 4 Establishment of real-time fluorescent quantitative PCR reaction system

qPCR nucleic acid amplification was performed using TransStart Probe qPCR SuperMix (Quanshijin Biotech). The reaction system was established according to Table 1, and the Rotor-Gene Q real-time fluorescent quantitative PCR instrument was used. The amplification conditions were pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 10 seconds, and extension at 60°C for 30 seconds. The result was judged as positive.

The establishment of embodiment 5 standard curve and repeatability check

Extract the plasmid (Omega Company) according to the instructions of the plasmid extraction kit, measure the concentration of the extracted plasmid with a UV spectrophotometer, and convert the concentration of the plasmid sample into the copy number concentration according to the molecular weight of the plasmid: the copy number of the detected gene in each µl sample = concentration ( ng/µl)×Avogadro’s constant×10 ^-9 /(660×number of recombinant plasmid bases). Use the dilution buffer (TransNGS ^TM Library Dilution Buffer of Quanshi Jin Biological Co., Ltd.) to dilute the above plasmids into 1.0×10 ² copies/µl ~ 1.0×10 ⁹ copies/µl, a total of 8 concentration gradients, each gradient standard 3 repetitions, negative control template with TE buffer, real-time fluorescent quantitative PCR detection.

When the recombinant plasmid concentration of the target gene is 6.91×10 ² copies/µl or above, the fluorescence signal intensity is higher than the detection threshold. When the recombinant plasmid concentration of the target gene was less than 6.91×10 ² copies/µl, no amplified fluorescent signal appeared, so the lower limit of detection of the recombinant plasmid containing the target gene was 6.91×10 ² copies/µl. In addition, in this PCR detection system, the concentration of the recombinant plasmid containing the B toxin gene is in the range of 10 ² to 10 ⁹ copies/µl, and there is a good linear relationship between the Ct value of the amplification reaction and the template concentration, as shown in Figure 1 and Figure 2 shows.

Embodiment 6 detection specificity evaluation

Method specificity evaluation, including the following steps:

(1) Sample source

Table 2 Experimental strains

serial number Bacteria name Latin name Strain qPCR 1 Clostridium botulinum B Clostridium botulinum Isolated strain + 1 Bacillus cereus Bacillus cereus Isolated strain - 2 Clostridium difficile Clostridium difficile Isolated strain - 3 Clostridium tertiary Clostridium tertium Isolated strain - 4 Clostridium perfringens Clostridium perfringens Isolated strain - 5 Clostridium sordarii Clostridium sordellii Isolated strain - 6 Enterobacter cloacae Enterobacter cloacae Isolated strain - 7 Streptococcus suis Streptococcus suis Isolated strain - 8 Yersinia enterocolitica Yersinia enterocxolitica ATCC23715 - 9 Enterococcus faecalis Enterococcus faecalis ATCC35667 - 10 Enterococcus faecium Enterococcus faecium Isolated strain - 11 Listeria monocytogenes Listeria monocytogenes Isolated strain - 12 Enterohemorrhagic Escherichia coli Enterohemorrhagic E. coli EDL933 - 13 Enteropathogenic Escherichia coli Enteropathogenic E. coli Isolated strain - 14 Enteroaggregative Escherichia coli Enteroaggregative E. coli Isolated strain - 15 Enterotoxigenic Escherichia coli Enterotoxigenic E. coli Isolated strain - 16 Enteroinvasive Escherichia coli Enteroinvasive E. coli Isolated strain - 17 Campylobacter jejuni Campylobacter jejuni ATCC33291 - 18 Vibrio cholerae Vibrio cholerae Isolated strain - 19 Salmonella typhi Salmonella typhi H98125 - 20 Salmonella choleraesuis Salmonella cholerae suis ATCC14028 - twenty one Citrobacter freundii Citrobacter freundii Isolated strain - twenty two Enterobacter sakazakii Enterobacter sakazakii ATCC51329 - twenty three Shigella flexneri Shigella flexneri Isolated strain - twenty four Shigella sonnei Shigella sonnei ATCC25931 - 25 Staphylococcus aureus Staphylococcus aureus ATCC6538 -

The isolated strains in Table 2 are all from the laboratory preservation bacteria of the Institute of Infectious Diseases Prevention and Control, Chinese Center for Disease Control and Prevention.

(2) Bacterial genomic DNA extraction;

Bacterial genomic DNA was extracted using a kit from Promega, operated according to the instructions, and stored at -20°C for future use.

(3) Specific detection test

The bacterial DNA obtained in step (1) and step (2) was used as a template, and water was used as a blank control to carry out qPCR reaction. The reaction system was established according to Table 1, and the Rotor-Gene Q real-time fluorescent quantitative PCR instrument was used, and the amplification condition was 95 Pre-denaturation at ℃ for 2min, denaturation at 95℃ for 10s, extension at 60℃ for 30s, a total of 45 cycles.

The results showed that the system was used against Bacillus cereus, Clostridium difficile, Clostridium third, Clostridium perfringens, Clostridium sordi, Enterobacter cloacae, Streptococcus suis, Yersinia enterocolitica, Enterococcus faecalis , Enterococcus faecium, Listeria monocytogenes, enterohemorrhagic Escherichia coli (EHEC), enteropathogenic Escherichia coli (EPEC), enteroaggregative Escherichia coli (EaggEC), enterotoxigenic Escherichia coli (ETEC), intestinal invasion coli (EIEC), Campylobacter jejuni, Vibrio cholerae, Salmonella typhi, Salmonella choleraesuis, Citrobacter freundii, Enterobacter sakazakii, Shigella flexneri, Shigella sonnei, Golden Genomic DNA of 25 kinds of pathogenic bacteria such as Staphylococcus aureus did not produce specific amplification curves when detected, indicating that the specificity of the detection system is good.

Example 7 Preparation and detection of simulated stool samples

Take healthy baby feces and pack 0.1 g/tube, 5 tubes in total. Add 100μlddH ₂ O to one of the tubes as a blank control, add 60μlddH ₂ O to the other four tubes, boil for 5 minutes, centrifuge at 13000rpm for 5 minutes, take the supernatant, and add 10 ³ to 10 ⁶ times diluted botulinum toxin to each tube in turn Clostridium recombinant plasmid standard solution 40μl. Three replicates were set for each concentration, and real-time fluorescent quantitative PCR detection was performed.

The results showed that when the recombinant plasmid concentration of the target gene was 2.1384×10 ³ copies/µl or above, the fluorescence signal intensity was higher than the detection threshold. When the concentration of the recombinant plasmid of the target gene was less than 2.1384×10 ³ copies/µl, there was no amplified fluorescent signal, so the lower limit of detection of the recombinant plasmid of the target gene in the simulated stool specimen was 2.1384×10 ³ copies/µl.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those skilled in the art, various changes or modifications can be made on the basis of the above description. All the implementation manners cannot be exhaustively listed here. All obvious changes or variations derived from the technical solutions of the present invention are still within the protection scope of the present invention.

sequence listing

<110> Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention

<120> Real-time fluorescent quantitative PCR nucleic acid sequence and kit for detecting Clostridium botulinum type B

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212>DNA

<213> Primers B-F (artificial sequence)

<400> 1

cacaaacatt gctagtgtaa ctgttaataa 30

<210> 2

<211> 29

<212>DNA

<213> Primer B-R (artificial sequence)

<400> 2

ctatagtctc attttcattt aaaactggc 29

<210> 3

<211> 32

<212>DNA

<213> Probe B-P (artificial sequence)

<400> 3

cagtaatcca ggagaagtgg agcgaaaaaa gg 32

Claims (10)

1. detecting the nucleic acid sequence of Type B clostridium botulinum, the nucleic acid sequence includes primer and probe, it is characterized in that the primer is The upstream primer B-F and downstream primer B-R of clostridium botulinum B toxin gene are detected, the probe is detection clostridium botulinum B toxin base The probe B-P of cause, upstream primer B-F, downstream primer B-R and probe B-P nucleotide sequence respectively as sequence SEQ ID No:1, Shown in SEQ ID No:2 and SEQ ID No:3, the end the 5' label reporter fluorescence group end JOE, the 3' label of the probe B-P is quenched Go out fluorophor BHQ2.

2. the nucleic acid sequence of detection Type B clostridium botulinum according to claim 1, it is characterized in that the target fragment of primer amplification Length is 130bp.

3. a kind of kit for detecting Type B clostridium botulinum, it is characterized in that the kit includes:

(1) qPCR reaction system composition, including upstream primer B-F, such as sequence SEQ as shown in sequence SEQ ID No:1 The end 5' of downstream primer B-R shown in ID No:2 and the probe B-P as shown in sequence SEQ ID No:3, the probe B-P mark Remember that the reporter fluorescence group end JOE, 3' marks quenching fluorescence group BHQ2；

(2) negative control；

(3) positive control.

4. kit according to claim 3, it is characterized in that the qPCR reaction system composition further includes qPCR SuperMix, the qPCR SuperMix contain dNTPs, Taq archaeal dna polymerase and PCR reaction buffer.

5. kit according to claim 3 or 4, it is characterized in that the negative control is TE buffer, TE buffer For 10mMTris-HCl, 1mM EDTA.

6. kit according to claim 3 or 4, it is characterized in that the positive control is Type B clostridium botulinum recombinant plasmid Standard items.

7. kit according to claim 3 or 4, it is characterized in that the kit further includes dilution buffer.

8. a kind of detection Type B clostridium botulinum type real-time fluorescence quantitative PCR detection method, it is characterized in that the following steps are included:

(1) sample to be tested bacterial genomes DNA is extracted；

(2) qPCR amplification is carried out to the sample DNA of extraction；Wherein use the upstream primer B- as shown in sequence SEQ ID No:1 F, the downstream primer B-R as shown in the sequence SEQ ID No:2 and probe B-P as shown in sequence SEQ ID No:3, the probe The end the 5' label reporter fluorescence group end JOE, 3' of B-P marks quenching fluorescence group BHQ2；

(3) PCR product is analyzed.

9. detection method according to claim 8, it is characterized in that 25 μ l of PCR reaction system is calculated as:

0.5 μ l of template DNA

10 μM of 0.5 μ l of primer B-F

10 μM of 0.5 μ l of primer B-R

10 μM of 0.5 μ l of probe B-P

2×qPCR SuperMix 12.5µl

ddH₂O 10.5µl。

10. detection method according to claim 8 or claim 9, it is characterized in that PCR reaction condition are as follows: after 95 DEG C of 2min, 95 DEG C of 10s, 60 DEG C of 30s mono- circulations, totally 45 recycle.