CN104962652A - wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei - Google Patents

wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei Download PDF

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CN104962652A
CN104962652A CN201510444097.XA CN201510444097A CN104962652A CN 104962652 A CN104962652 A CN 104962652A CN 201510444097 A CN201510444097 A CN 201510444097A CN 104962652 A CN104962652 A CN 104962652A
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王海艳
敖威华
赵治国
张捷
催强
赵林立
李刚
潘国卿
韩祎陟
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INNERMONGOLIA ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
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Abstract

The invention discloses a wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei. The invention firstly discloses a real-time fluorescent PCR primer pair and probe for identifying Shigella sonnei, wherein the primer pair is composed of a forward primer of which the nucleotide sequence is disclosed as SEQ ID No.1 and a reverse primer of which the nucleotide sequence is disclosed as SEQ ID No.2; and the nucleotide sequence of the probe is disclosed as SEQ ID No.3. The invention also discloses a real-time fluorescent PCR method for identifying Shigella sonnei, which comprises the following steps: extracting DNA of the sample to be detected as a template, establishing a PCR reaction system by using the primer pair and probe, and carrying out PCR amplification; and if an S-shaped amplification curve appears in the result, determining that the sample to be detected contains Shigella sonnei. The real-time fluorescent PCR method has the advantages of high sensitivity and time saving, is convenient and quick, and can be used for detecting Shigella sonnei in samples.

Description

Real-time fluorescent PCR method and kit for identifying Shigella sonnei aiming at wbgZ gene
Technical Field
The invention relates to a method for detecting Shigella sonnei, in particular to a real-time fluorescent PCR method for identifying Shigella sonnei aiming at a wbgZ gene, and also relates to a real-time fluorescent PCR detection primer and a kit for the Shigella sonnei, belonging to the field of detection of the Shigella sonnei.
Background
Shigella still causes major health problems in many countries and regions of the world, and particularly in underdeveloped and developing countries, the common health problems caused by Shigella are more serious due to a lack of clean water sources, lack of necessary sanitary and medical equipment and the like. Worldwide, shigellasis is the most prevalent in children 1-5 years of age or older and has the highest incidence and mortality. Most shigellosis is associated with three shigellosis, namely shigella flexneri, shigella sonnei and shigella dysenteriae. Of the three shigellas, shigella sonnei is found mainly in industrialized countries, shigella flexneri is found mainly in developing countries, and shigella dysenteriae is the only strain that causes epidemic and pandemic shigella.
At present, most Shigella can not be identified and grouped due to the lack of accurate and reliable identification technology in developing countries, and the identification and grouping of the Shigella mainly depend on conventional biochemical identification methods and immunological methods which are relatively complex, time-consuming, labor-consuming and low in sensitivity. Some researchers also research and establish molecular biology detection technologies to mainly identify Shigella, but certain limitations also exist to cause false detection and missed detection. Therefore, it is very important to establish an accurate, reliable and fast shigella identification grouping technology.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer pair and a probe for real-time fluorescent PCR detection of Shigella sonnei;
the invention aims to solve another technical problem of providing a real-time fluorescence PCR method for identifying Shigella sonnei;
the third technical problem to be solved by the invention is to provide a real-time fluorescence PCR detection kit for Shigella sonnei.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention designs primers and probes according to the wbgZ gene of Shigella sonnei. The invention firstly discloses a primer pair and a probe of real-time fluorescence PCR for identifying Shigella sonnei aiming at wbgZ gene, wherein the primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.1 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 2; the nucleotide sequence of the probe is shown as SEQ ID No. 3; or,
the primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.4 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 5; the nucleotide sequence of the probe is shown in SEQ ID No. 6; or,
the primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.7 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 8; the nucleotide sequence of the probe is shown in SEQ ID No. 9.
The invention also designs 3 pairs of primers and probes as internal references according to ompA gene, wherein the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.10 and a downstream primer with a nucleotide sequence shown as SEQ ID No.11, and the nucleotide sequence of the probe is shown as SEQ ID No. 12; or the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.13 and a downstream primer with a nucleotide sequence shown as SEQ ID No.14, and the nucleotide sequence of the probe is shown as SEQ ID No. 15; or the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.16 and a downstream primer with a nucleotide sequence shown as SEQ ID No.17, and the nucleotide sequence of the probe is shown as SEQ ID No. 18.
The invention uses 17 Shigella and 19 non-Shigella to carry out specificity experiments on the primers and probes used by the real-time fluorescent PCR method of Shigella sonnei, and the results show that the primer pairs and the probes Son1-F/Son1-R/Son1-P (namely the primer pairs consisting of SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequence of the probe is shown in SEQ ID No. 3) have good specificity, an amplification curve is only shown in Shigella sonnei, and no amplification curve is shown in other Shigella (such as Shigella flexneri, Shigella poensis and Shigella dysenteriae) and non-Shigella. The internal reference primer and the probe Ctr1-F/Ctr1-R/Ctr1-P (namely a primer pair consisting of SEQ ID No.10 and SEQ ID No. 11; the nucleotide sequence of the probe is shown as SEQ ID No. 12) can generate an amplification curve in all bacteria.
While the primer and probe Son2-F/Son2-R/Son2-P (i.e. the primer pair consisting of SEQ ID No.4 and SEQ ID No. 5; the nucleotide sequence of the probe is shown as SEQ ID No. 6), Son3-F/Son3-R/Son3-P (i.e. the primer pair consisting of SEQ ID No.7 and SEQ ID No. 8; the nucleotide sequence of the probe is shown as SEQ ID No. 9), internal reference primer and probe Ctr2-F/Ctr2-R/Ctr2-P (i.e. the primer pair consisting of SEQ ID No.13 and SEQ ID No. 14; the nucleotide sequence of the probe is shown as SEQ ID No. 15), the real-time fluorescent PCR specificity of Ctr3-F/Ctr3-R/Ctr3-P (namely a primer pair consisting of SEQ ID No.16 and SEQ ID No. 17; the nucleotide sequence of the probe is shown as SEQ ID No. 18) is not good.
Therefore, the primer and the probe Son1-F/Son1-R/Son1-P (namely a primer pair consisting of SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequence of the probe is shown in SEQ ID No. 3) are selected for identifying the Shigella sonnei, and the primer and the probe Ctr1-F/Ctr1-R/Ctr1-P are used as internal references (namely a primer pair consisting of SEQ ID No.10 and SEQ ID No. 11; the nucleotide sequence of the probe is shown in SEQ ID No. 12).
The primer pair and the probe can be applied to identification of Shigella sonnei.
The invention further discloses a real-time fluorescence PCR method for identifying Shigella sonnei aiming at the wbgZ gene, which comprises the following steps: (1) extracting DNA of a sample to be detected; (2) establishing a PCR reaction system by using the extracted DNA as a template and the primer pair and the probe, and carrying out real-time fluorescence PCR amplification; (3) if an S-shaped amplification curve appears in the real-time fluorescent PCR amplification result, judging that the sample to be detected contains Shigella sonnei; and if the real-time fluorescent PCR amplification result does not have an S-shaped amplification curve, judging that the sample to be detected does not contain Shigella sonnei.
The total volume of the PCR reaction system was 25. mu.L, wherein FastStart Universal Probe Master was 12.5. mu.L, 10 pmol/. mu.L of the upstream primer was 1.0. mu.L, 10 pmol/. mu.L of the downstream primer was 1.0. mu.L, 10 pmol/. mu.L of the probe was 1.0. mu.L, 20. mu.g/. mu.L of the template DNA was 2.0. mu.L, and the balance was sterilized distilled water.
The nucleotide sequence of the upstream primer is shown as SEQ ID No.1, the nucleotide sequence of the downstream primer is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. The 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end of the probe is marked with a fluorescence quenching group; preferably, the fluorescence reporter group is a FAM fluorescence reporter group, and the fluorescence quencher group is a TAMRA fluorescence quencher group.
The real-time fluorescent PCR amplification conditions are as follows: 3min at 95 ℃; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, 40 cycles. In addition, while a sample is amplified, an internal reference primer and a probe are used for amplification, the annealing temperature of the internal reference primer and the probe Ctr1-F/Ctr1-R/Ctr1-P (namely a primer pair consisting of SEQ ID No.10 and SEQ ID No. 11; the nucleotide sequence of the probe is shown in SEQ ID No. 12) is 57 ℃, and the rest of amplification conditions and a PCR reaction system are the same as the primer and the probe Son1-F/Son1-R/Son 1-P.
The real-time fluorescent PCR sensitivity test result shows that the minimum detection limit of PCR is 1-3 cfu/25g (mL) after the shigella sonnei is subjected to two-step enrichment and PCR detection, and the real-time fluorescent PCR method has extremely high sensitivity.
In order to improve the detection sensitivity, the invention adopts a bacterium increasing step before the real-time fluorescent PCR detection, and has the advantages that: the damaged cells are repaired, the number of the cells is increased, and the inhibitor and dead cells in the sample are diluted, so that false negative or false positive is avoided, the detection sensitivity is increased, and the detection reliability is improved. The result of the invention shows that after enrichment, real-time fluorescence PCR detection is directly carried out on the enriched broth, the detection sensitivity can reach 1-3 cfu/25g (mL) in other foods with slight mixed bacteria pollution, and the detection limits respectively reach less than or equal to 8cfu/25mL, 12cfu/25g and less than or equal to 12cfu/25g even in raw milk, raw meat and milk powder with a large amount of mixed bacteria interference. Therefore, the enrichment step is essential to improve the sensitivity of detection. In addition, the enrichment broth is inoculated to a solid culture medium for separation culture by streaking, and then the suspicious bacterial colony is subjected to real-time fluorescence PCR identification, so that the result can detect the Shigella sonnei in all detection samples, the detection limit of the method is consistent with that of the GB4789.5 traditional culture method and reaches 1-3 cfu/25g (mL), and the detection sensitivity of the method reaches the detection level of the traditional culture method, and the method can be used for the conventional detection and analysis of the Shigella sonnei in the sample.
According to the anti-interference test result, the Shigella sonnei can be detected by the real-time fluorescent PCR method and the culture method under the condition that the interference bacteria are artificially polluted, but the culture method is relatively labor-consuming. In the presence of bacteria of the genus other than Shigella, the growth of the bacteria of the genus other than Shigella is inhibited by adding the bacteriostatic agent to the enrichment broth, so that the target bacteria can be easily detected by both methods.
The actual sample detection result shows that 1100 samples which are subjected to detection are detected by adopting the real-time fluorescent PCR method, and compared with the GB4789.5 traditional culture method, the results are not separated into Shigella sonnei, and the detection result is consistent with that of the GB4789.5 traditional culture method.
The invention also discloses a real-time fluorescence PCR detection kit for Shigella sonnei, which comprises: FastStart Universal Probe Master, amplification primer pair, Probe and sterilized distilled water; wherein the amplification primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.1 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 2; the nucleotide sequence of the probe is shown in SEQ ID No. 3. Wherein, the 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group; preferably, the fluorescence reporter group is a FAM fluorescence reporter group, and the fluorescence quencher group is a TAMRA fluorescence quencher group.
The real-time fluorescent PCR detection kit of the invention also comprises: an internal reference primer pair and a probe; preferably, the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.10 and a downstream primer with a nucleotide sequence shown as SEQ ID No.11, and the nucleotide sequence of the probe is shown as SEQ ID No. 12; or the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.13 and a downstream primer with a nucleotide sequence shown as SEQ ID No.14, and the nucleotide sequence of the probe is shown as SEQ ID No. 15; or the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.16 and a downstream primer with a nucleotide sequence shown as SEQ ID No.17, and the nucleotide sequence of the probe is shown as SEQ ID No. 18. Wherein the annealing temperature of the internal reference primer pair is 57 ℃; the 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end of the probe is marked with a fluorescence quenching group; preferably, the fluorescence reporter group is a FAM fluorescence reporter group, and the fluorescence quencher group is a TAMRA fluorescence quencher group.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the invention designs primers and probes for real-time fluorescent PCR analysis according to the wbgZ gene of Shigella sonnei. The real-time fluorescent PCR method is compared with the traditional culture method, and the result shows that the real-time fluorescent PCR method has the same detection level as the traditional culture method, but the real-time fluorescent PCR method is more time-saving, convenient and quick. The specific experiment result shows that the Son1-F/Son1-R/Son1-P has a specific fluorescence curve in the amplification of Shigella sonnei, and other groups of Shigella and non-Shigella do not have specific fluorescence curves, so that the method can be used for detecting the Shigella sonnei in a sample.
Drawings
FIG. 1 shows the amplification results of the primer and probe Son1-F/Son1-R/Son 1-P; wherein the amplification curves are the results of Shigella sonnei CMCC51592 strain, Shigella sonnei ATCC11060 strain, Shigella sonnei ATCC29930 strain, Shigella sonnei ATCC25931 strain and Shigella sonnei CMCC51334 strain respectively; at baseline, the strains are Shigella flexneri CMCC51571, Shigella flexneri CMCC51572, Shigella flexneri ATCC12022, Shigella flexneri CMCC51579, Shigella flexneri CMCC51240, Shigella boyneri CMCC51346, Shigella boyneri CMCC51585, Shigella boyneri CMCC51586, Shigella boyneri ATCC9207, Shigella dysenteriae CMCC51105, Shigella dysenteriae CMCC51570, Shigella dysenteriae CMCC51252, Listeria monocytogenes CMCC54002, Listeria Ennokogyl ATCC33090, Listeria vachelli ATCC35897, Listeria schnei ATCC35967, Listeria glazierii ATCC25401, Bacillus CMCC63501-21, Escherichia coli 44116, Salmonella saxosa 50079, Staphylococcus aureus type Staphylococcus aureus CGCC 26001, Bacillus subtilis ATCC 359610, Escherichia coli CMCC 294847, Escherichia coli CMCC 2947, Salmonella sakazakii CMCC 51544, and Salmonella sakazakii CMCC 29303, Pseudomonas aeruginosa ATCC27853 strain, yersinia enterocolitica ATCC23715 strain, listeria ovis ATCC19119, escherichia coli O157: h7ATCC43895 strain and blank control results;
FIG. 2 shows the amplification results of primers and probes Ctr1-F/Ctr1-R/Ctr 1-P; wherein the amplification curves are respectively Shigella flexneri CMCC51571 strain, Shigella flexneri CMCC51572 strain, Shigella flexneri ATCC12022 strain, Shigella flexneri CMCC51579 strain, Shigella flexneri CMCC51240 strain, Shigella boyneri CMCC51346 strain, Shigella boyneri CMCC51585 strain, Shigella boyneri CMCC51586 strain, Shigella boyneri ATCC9207 strain, Shigella sonnei CMCC51592 strain, Shigella sonnei ATCC11060 strain, Shigella sonnei ATCC29930 strain, Shigella sonnei ATCC25931 strain, Shigella sonnei CMCC51334 strain, Shigella dysenteriae CC51105 strain, Shigella dysenteriae CMCC51570 strain, Shigella dysenteriae CMCC51252 strain, Listeria monocytogenes 54002 strain, Salmonella saxolisurensis ATCC33090 strain, Weisse wel welfare 35897 strain, ATCC35897 strain, Staphylococcus aureus CMCC 44401 strain, Staphylococcus aureus CMCC 44501 strain, Staphylococcus aureus CMCC 500401 strain, Staphylococcus aureus strain CMCC 51501 strain, Salmonella choleraesu, Klebsiella pneumoniae CMCC46114 strain, Citrobacter freundii CMCC48001 strain, Enterobacter sakazakii ATCC29544 strain, Bacillus cereus CMCC63303 strain, Enterobacter cloacae ATCC13047 strain, Pseudomonas aeruginosa ATCC27853 strain, Yersinia enterocolitica ATCC23715 strain, Listeria ovis ATCC19119, Escherichia coli O157: results of H7ATCC43895 strain; blank control results at baseline position;
FIG. 3 shows the amplification results of the primer and probe Son1-F/Son1-R/Son 1-P; wherein the amplification curves are the results of the inoculation amounts of 100cfu/25g (mL), 50cfu/25g (mL), 25cfu/25g (mL), 10cfu/25g (mL), 5cfu/25g (mL) and 1-3 cfu/25g (mL), respectively; blank control results at baseline position;
FIG. 4 shows the amplification results of primers and probes Ctr1-F/Ctr1-R/Ctr 1-P; wherein the amplification curves are the results of the inoculation amounts of 100cfu/25g (mL), 50cfu/25g (mL), 25cfu/25g (mL), 10cfu/25g (mL), 5cfu/25g (mL) and 1-3 cfu/25g (mL), respectively; blank control results are at baseline.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
1. Material
1.1 Primary reagents
Shigella enrichment broth and Shigella chromogenic medium are purchased from Beijing land bridge company; TrisBase, EDTA, lysozyme, guanidinium isothiocyanate, sarkosyl were purchased from Dalibao Bio; the fluorescent PCR reaction solution FastStart Universal Probe Master (Rox) a was purchased from Roche, and all other reagents were domestic analytical reagents.
1.2 Main instrumentation
A constant temperature incubator, a vortex oscillator, an electronic balance, a low-temperature freezing high-speed centrifuge, a micropipettor, an LC480 type real-time fluorescence PCR instrument and the like.
1.3 bacterial strains
Shigella and other bacteria of the genus Shigella were purchased from the Chinese pharmaceutical biologies institute and Hanni Shanghai.
1.4 samples
Sterilizing fresh milk, ice cream, cheese, yogurt, sausage, cooked meat and raw meat, and purchasing in supermarket; the raw milk is purchased from individual cattle farms, and the milk powder is a sample to be checked.
Example 1 establishment of real-time fluorescence PCR method for identifying Shigella sonnei
1. Materials and methods
1.1 primers and probes
According to the Shigella sonnei wbgZ gene, 3 pairs of primers and probes are designed by utilizing an http:// biolnfo. ut. ee/primer 3-0.4.0/primer online design website for identifying the Shigella sonnei, 3 pairs of primers and probes are designed according to ompA gene as internal references, the primers and probes are synthesized by Ogil biotechnology Limited in Shanghai, and the sequences are shown in Table 1.
TABLE 1 primers and probes
1.2 methods
1.2.1 real-time fluorescent PCR specificity detection
1.2.1.1 bacterial culture
All bacteria (Table 2) were inoculated into TSB-YE and incubated at 37 ℃ for 24 h. Respectively taking 1.5mL of bacterial culture fluid, centrifuging for 5min at 8000r/min, discarding the supernatant, washing the precipitate with 0.5mL of TE once, and directly using for DNA extraction or storing at-20 ℃ for later use.
1.2.1.2 DNA extraction (guanidinium isothiocyanate extraction)
1mL of the bacterial suspension was added to a 1.5mL reaction tube, centrifuged at 8000g for 5min, and the supernatant was discarded.
The pellet was resuspended in 0.5mL TE buffer, centrifuged at 8000g for 5min, and the supernatant discarded.
For gram-positive bacteria, the pellet was resuspended in 100. mu.L of lysozyme solution and bathed in a 37 ℃ water bath for 30 min. (for gram-negative bacteria, after directly using 100 u L TE buffer solution heavy suspension, continue the next step.)
The reaction tube was rapidly cooled by displacement into an ice bath.
0.5mL of guanidinium isothiocyanate solution was added and allowed to act at room temperature for 10min, during which time the tube wall was flicked.
The reaction tube was placed on ice, 0.25mL of 7.5M ammonium acetate solution was added, mixed well and ice-cooled for 10 min.
0.5mL of phenol/chloroform/isoamyl alcohol solution (25: 24: 1) was added thereto, and after mixing well, the mixture was centrifuged at 12000g for 10 min.
Transferring the supernatant into a new reaction tube, adding 0.54 volume of cold isopropanol, mixing, and standing at-20 deg.C for 10 min.
The supernatant was discarded, and the precipitate was washed once with 75% ethanol and dried under vacuum.
Dissolving the precipitate with 50-60 μ L sterilized double distilled water, and using immediately or storing at-20 deg.C for later use.
1.2.1.3 real-time fluorescent PCR amplification
Using the extracted bacterial DNA as a template, and respectively using each pair of primers and probes to carry out PCR amplification, wherein the reaction system is 25 mu L:
the reaction conditions with the LC480 type real-time fluorescent PCR instrument were: 3min at 95 ℃; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s (annealing temperature of internal reference primer and probe is 57 ℃), and reaction conditions can be changed according to the fluorescence PCR instrument used for 40 cycles.
2. Results of the experiment
2.1 determination of the specificity of real-time fluorescent PCR
The result shows that the primer and the probe Son1-F/Son1-R/Son1-P have good specificity, an amplification curve only appears in Shigella sonnei, and no amplification curve appears in other Shigella (such as Shigella flexneri, Shigella boydii and Shigella dysenteriae) and non-Shigella, and the result is shown in Table 2 and figure 1.
The primers and the probe Ctr1-F/Ctr1-R/Ctr1-P were able to show an amplification curve in all bacteria, and the results are shown in Table 2 and FIG. 2.
The primer and the probe Son2-F/Son2-R/Son2-P, Son3-F/Son3-R/Son3-P have poor specificity of real-time fluorescent PCR, and other Shigella bacteria and non-Shigella bacteria except for Shigella sonnei also have amplification curves; the primers and the probe Ctr2-F/Ctr2-R/Ctr2-P, Ctr3-F/Ctr3-R/Ctr3-P can not generate an amplification curve in all bacteria, and the specificity is not good.
Therefore, the primer and the probe Son1-F/Son1-R/Son1-P are selected for identifying the Shigella sonnei, and the primer and the probe Ctr1-F/Ctr1-R/Ctr1-P are used as internal references.
TABLE 2 real-time fluorescent PCR amplification results for primers and probes
Example 2 real-time fluorescent PCR sensitivity assay and actual sample detection for identification of Shigella sonnei
The invention selects a primer and a probe Son1-F/Son1-R/Son1-P (namely a primer pair consisting of SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequence of the probe is shown in SEQ ID No. 3) for identifying Shigella sonnei, and takes the primer and the probe Ctr1-F/Ctr1-R/Ctr1-P (namely a primer pair consisting of SEQ ID No.10 and SEQ ID No. 11; the nucleotide sequence of the probe is shown in SEQ ID No. 12) as an internal reference.
1. Experimental methods
1.1 real-time fluorescent PCR sensitivity test
1.1.1 bacterial strains
Shigella and other bacteria were purchased from the Chinese pharmaceutical biologies institute and Hanny Shanghai, Inc., and the strains are shown in Table 2.
1.1.2 samples
Sterilizing fresh milk, ice cream, cheese, yogurt, sausage, cooked meat and raw meat, and purchasing in supermarket; the raw milk is purchased from individual cattle farms, and the milk powder is a sample to be checked.
1.1.3 quantitative inoculation and culture of bacteria
The method comprises inoculating Shigella sonnei strains into 225mL of Shigella enrichment broth added with 25mL of sterilized normal saline, respectively, in amounts of 1-3 cfu/25g (mL), 5cfu/25g (mL), 10cfu/25g (mL), 25cfu/25g (mL), 50cfu/25g (mL) and 100cfu/25g (mL), and performing anaerobic culture at 41.5 ℃ for 18h with a negative control without adding bacteria. 1mL of each culture was centrifuged at 8000r/min for 5min, the pellet was suspended in 0.5mL of TE buffer (pH8.0), transferred to a 1.5mL centrifuge tube, centrifuged at 8000r/min for 5min, and the supernatant was discarded and used directly for DNA extraction or stored in a freezer at-20 ℃ for further use.
The method of DNA extraction and real-time fluorescent PCR amplification was the same as in example 1.
1.2 anti-interference test
Inoculating the mixture of Shigella sonnei and non-Shigella bacteria type B hemolytic streptococcus into Shigella enrichment broth (the plates of Shigella chromogenic culture medium are used for counting while inoculating), wherein the inoculum size is shown in Table 3, and the culture, nucleic acid extraction and real-time fluorescence PCR amplification test are carried out according to the method 1.2 in the embodiment 1; and meanwhile, a GB4789.5 traditional culture method is adopted for comparison test.
1.3 verification test of artificially inoculated samples
5 samples of sterilized fresh milk, ice cream, yoghourt, cheese, sausage, cooked meat, raw milk and milk powder are respectively taken, and the samples are used as an artificial inoculation experiment of a real-time fluorescence PCR detection method after no Shigella contamination is detected by a conventional method.
Adding 25g (mL) of each sample into 225mL of Shigella enrichment broth, homogenizing, performing artificial inoculation test according to the bacterial amount in Table 4, and performing transfer, culture, DNA extraction and real-time fluorescence PCR amplification according to the method 1.2 in example 1; meanwhile, the enrichment broth is streaked and inoculated to a flat plate of a shigella chromogenic culture medium for isolated culture, and then suspicious colonies are picked for real-time fluorescent PCR identification; and a comparative experiment was carried out using the conventional culture method of GB 4789.5.
1.4 actual sample detection
Detecting 1100 samples which are submitted for inspection in the laboratory and purchased in the market; and compared with the conventional culture method of GB 4789.5.
2. Results of the experiment
2.1 determination of real-time fluorescent PCR sensitivity
The method comprises the steps of using physiological saline to replace food, respectively and quantitatively inoculating Shigella sonnei into Shigella enrichment broth, carrying out PCR detection after two steps of enrichment, wherein the minimum detection limit of PCR is 1-3 cfu/25g (mL) after enrichment, the PCR amplification result of a primer and a probe Son1-F/Son1-R/Son1-P is shown in figure 3, and the PCR amplification result of the primer and the probe Ctr1-F/Ctr1-R/Ctr1-P is shown in figure 4. The result proves that the real-time fluorescence PCR method has extremely high sensitivity.
2.2 anti-interference test results
According to tests, the real-time fluorescent PCR method and the culture method established by the invention can detect the Shigella sonnei under the condition of artificial pollution of interfering bacteria, but the culture method is relatively laborious. In the presence of bacteria of the genus other than Shigella, since the growth of bacteria of the genus other than Shigella was inhibited by adding the bacteriostatic agent to the enrichment broth, the target bacteria were easily detected by both methods, and the results are shown in Table 3.
TABLE 3 Shigella sonnei test results for anti-beta hemolytic streptococcal interference
2.3 detection of Shigella sonnei in artificially contaminated samples
After the artificial contaminated sample is subjected to enrichment for 18 hours at the temperature of 41.5 ℃, the enrichment broth is directly subjected to real-time fluorescence PCR analysis, and as a result, the detection limit of Shigella sonnei in disinfected milk, ice cream, yoghourt, cheese, cooked meat and sausages is 1-3 cfu/25g (mL); the detection limits in the raw milk, the raw meat and the milk powder are respectively less than or equal to 8cfu/25mL, 12cfu/25g and less than or equal to 12cfu/25 g. The enrichment broth is inoculated to a solid culture medium for separation culture by streaking and then subjected to real-time fluorescence PCR analysis, the result shows that the Shigella sonnei can be detected in all samples, the detection limit is consistent with that of the GB4789.5 traditional culture method, the detection limit reaches 1-3 cfu/25g (mL), and the result is shown in Table 4. The real-time fluorescence PCR method is proved to be suitable for routine detection and analysis of different types of samples.
TABLE 4 detection of Shigella sonnei in artificially contaminated samples
Note: directly taking the parts of PCR positive samples detected by enrichment broth; b, separating the positive sample parts detected by PCR after culture; c, the number of positive samples detected by the traditional culture method of GB 4789.5; d, inoculating the sample parts of the Shigella sonnei.
2.4 actual sample detection
The real-time fluorescent PCR method established by the invention is used for detecting 1100 samples which are sent for detection, and compared with the GB4789.5 traditional culture method, the results are not separated into Shigella sonnei, and the detection results are consistent with those of the GB4789.5 traditional culture method.

Claims (10)

1. The primer pair and the probe of the real-time fluorescence PCR for identifying the Shigella sonnei aiming at the wbgZ gene are characterized in that: the primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.1 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 2; the nucleotide sequence of the probe is shown as SEQ ID No. 3; or,
the primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.4 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 5; the nucleotide sequence of the probe is shown in SEQ ID No. 6; or,
the primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.7 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 8; the nucleotide sequence of the probe is shown in SEQ ID No. 9.
2. The primer pair and the probe of claim 1 are applied to identification of Shigella sonnei.
3. A real-time fluorescence PCR method for identifying Shigella sonnei aiming at wbgZ gene is characterized by comprising the following steps: (1) extracting DNA of a sample to be detected; (2) establishing a PCR reaction system by using the extracted DNA as a template and the primer pair and the probe of claim 1 for real-time fluorescent PCR amplification; (3) if an S-shaped amplification curve appears in the real-time fluorescent PCR amplification result, judging that the sample to be detected contains Shigella sonnei; and if the real-time fluorescent PCR amplification result does not have an S-shaped amplification curve, judging that the sample to be detected does not contain Shigella sonnei.
4. The real-time fluorescent PCR method of claim 3, wherein: the total volume of the PCR reaction system is 25 mu L; wherein, FastStart Universal Probe Master 12.5. mu.L, 10 pmol/. mu.L upstream primer 1.0. mu.L, 10 pmol/. mu.L downstream primer 1.0. mu.L, 10 pmol/. mu.L Probe 1.0. mu.L, 20. mu.g/. mu.L template DNA 2.0. mu.L, and the balance of sterilized distilled water.
5. The real-time fluorescent PCR method of claim 4, wherein: the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, the nucleotide sequence of the downstream primer is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
6. The real-time fluorescent PCR method according to claim 3, 4 or 5, characterized in that: the 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end of the probe is marked with a fluorescence quenching group;
preferably, the fluorescence reporter group is a FAM fluorescence reporter group, and the fluorescence quencher group is a TAMRA fluorescence quencher group.
7. The real-time fluorescent PCR method of claim 3, wherein: the real-time fluorescent PCR amplification conditions are as follows: 3min at 95 ℃; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, 40 cycles.
8. A real-time fluorescence PCR detection kit for Shigella sonnei comprises: FastStartUniversal Probe Master, amplification primer pair, Probe and sterilized distilled water; the method is characterized in that: the amplification primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.1 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 2; the nucleotide sequence of the probe is shown as SEQ ID No. 3.
9. The real-time fluorescent PCR detection kit according to claim 8, further comprising: an internal reference primer pair and a probe;
preferably, the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.10 and a downstream primer with a nucleotide sequence shown as SEQ ID No.11, and the nucleotide sequence of the probe is shown as SEQ ID No. 12; or,
the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.13 and a downstream primer with a nucleotide sequence shown as SEQ ID No.14, and the nucleotide sequence of the probe is shown as SEQ ID No. 15; or,
the internal reference primer pair consists of an upstream primer with a nucleotide sequence shown as SEQ ID No.16 and a downstream primer with a nucleotide sequence shown as SEQ ID No.17, and the nucleotide sequence of the probe is shown as SEQ ID No. 18;
more preferably, the annealing temperature of the internal reference primer pair is 57 ℃.
10. The real-time fluorescent PCR detection kit according to claim 8 or 9, characterized in that: the 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end of the probe is marked with a fluorescence quenching group;
preferably, the fluorescence reporter group is a FAM fluorescence reporter group, and the fluorescence quencher group is a TAMRA fluorescence quencher group.
CN201510444097.XA 2015-07-27 2015-07-27 wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei Pending CN104962652A (en)

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CN101440404A (en) * 2008-11-27 2009-05-27 中国人民解放军军事医学科学院微生物流行病研究所 Method for detecting Shigella sonnei by using suspension chip technology
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CN101440404A (en) * 2008-11-27 2009-05-27 中国人民解放军军事医学科学院微生物流行病研究所 Method for detecting Shigella sonnei by using suspension chip technology
CN103451307A (en) * 2013-09-17 2013-12-18 北京卓诚惠生生物科技有限公司 Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit

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