CN106434901B - method for detecting six kinds of diarrhea escherichia coli and shigella by multiplex PCR - Google Patents
method for detecting six kinds of diarrhea escherichia coli and shigella by multiplex PCR Download PDFInfo
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Abstract
The invention provides a primer group for detecting six kinds of Escherichia coli and Shigella diarrhoea by multiplex PCR, wherein the primer group comprises primers shown in SEQ ID NO.1-29, and the invention also provides a detection method and a kit for the six kinds of Escherichia coli and Shigella diarrhoea. Through the technical scheme, the invention provides a complete technical means for quickly and accurately screening the six kinds of Escherichia coli and Shigella diarrhoea, can realize quick screening and identification of the six kinds of Escherichia coli and Shigella diarrhoea after a food poisoning event occurs and after epidemic outbreak, and provides a reliable basis for reasonably and properly treating the epidemic situation.
Description
Technical Field
the invention relates to the field of biotechnology, in particular to a primer group and a kit for detecting six kinds of Escherichia coli and Shigella diarrhoea by multiplex PCR and a detection method thereof.
Background
Shigella is a gram-negative intestinal pathogenic bacterium with high infectivity and serious harm, and Shigella dysentery (shigelosis) caused by clinical infection is one of important infectious diseases in developing countries and is also a main pathogen of diarrhea in developed countries.
Escherichia coli (Escherichia coli) is an important normal flora in the intestinal tract. But can become conditioned pathogenic bacteria after the host immunity is reduced or bacteria invade tissues and organs outside the intestinal tract, thereby causing the infection outside the intestinal tract. Secondly, some strains of Escherichia coli carry pathogenic genes and cause diarrhea, and are known as diarrheagenic Escherichia coli. The diarrhea causing escherichia coli is known to be: enteroinvasive escherichia coli (EIEC), enterotoxigenic escherichia e.coli (ETEC), enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (EHEC), enteroaggregative escherichia coli (EAEC), and enterodiffusible escherichia coli (DAEC).
The main methods adopted by China to detect the Escherichia coli and Shigella diarrhoea at present are respectively based on national standards GB/T4789.6-2003 and GB/T4789.5-2012, and the detection process comprises the steps of sample acquisition, enrichment, isolated culture, gram stainboscopy/biochemistry and colony observation/biochemical reaction experiments, enterotoxin detection/serology identification. The whole detection process is complex in operation, long in time, requires about 4-7 days, is time-consuming and labor-consuming, has a single detection target, is limited by factors such as limited database information, and the obtained detection data are unreliable.
The molecular biology detection method provides a good detection tool for detecting the Escherichia coli and the Shigella, and a technology for rapidly detecting the Escherichia coli and the Shigella by adopting a PCR technology is established at home and abroad at present. However, the single PCR technology is mostly used for detection, the annealing temperatures and the concentrations of a plurality of pairs of primers are different, multiple PCR reactions are needed to be detected one by one, the method is complex to operate, time and labor are consumed, and the quality control of each reaction system needs to be well done.
The existing scheme for detecting the Escherichia coli and the Shigella by using the multiplex PCR method cannot well distinguish the diarrheagenic Escherichia coli from the non-diarrheagenic Escherichia coli and cannot accurately judge the pathotype of the Escherichia coli. The document 'a multiple PCR method for rapidly identifying 7 common intestinal pathogens' reports a technical scheme for simultaneously detecting 7 common intestinal pathogens. However, the difference between the estIa and estIb gene amplification fragments of the method is only 15bp, and the estIa and estIb gene amplification fragments cannot be distinguished by agarose electrophoresis of less than 2.5 percent; the salmonella invA gene amplification product and the stx1A gene amplification fragment cannot be distinguished, and need to be preliminarily distinguished according to the characteristics of colonies on a selective plate; false negative results cannot be effectively identified because of no amplification internal standard; meanwhile, the method has the defects of insufficient diarrhea type coverage of virulence genes and general sensitivity.
Therefore, there is a need to provide a new technical scheme for simultaneously carrying out rapid, specific and sensitive detection on six pathogenic Escherichia coli and Shigella.
disclosure of Invention
The invention aims to solve the problems in the current technology for simultaneously detecting six pathogenic types of Escherichia coli, namely EPEC, EHEC, ETEC, EAEC, DAEC, EIEC and Shigella, and provides a novel technical scheme capable of simultaneously carrying out rapid, specific and sensitive detection on the six pathogenic types of Escherichia coli and the Shigella.
In order to achieve the above purposes, the invention provides a primer group for detecting six kinds of Escherichia coli and Shigella diarrhoea by multiplex PCR, wherein the primer group comprises primers shown in SEQ ID NO. 1-29; the six diarrheagenic Escherichia coli include enteroinvasive Escherichia coli (EIEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enteroaggregative Escherichia coli (EAEC), and diffusible Escherichia coli (DAEC).
the invention also provides a detection method of six kinds of diarrhea Escherichia coli and Shigella, which comprises the following steps:
(1) Extracting the total DNA of a sample to be detected;
(2) Carrying out multiple PCR reaction by using the total DNA as a template and the primer group to obtain a material subjected to multiple PCR amplification;
(3) And carrying out nucleic acid electrophoresis detection on the material subjected to the multiple PCR amplification to obtain a nucleic acid electrophoresis detection result.
The invention also provides a kit for detecting six kinds of diarrhea escherichia coli and shigella by multiplex PCR, and the kit comprises the primer group and the DNA polymerase.
on the other hand, the invention also provides the application of the primer group in the preparation of a kit for detecting six kinds of Escherichia coli and Shigella dysenteriae; wherein the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible Escherichia coli.
the six kinds of diarrhea Escherichia coli and Shigella identification multiplex PCR detection method established by the invention can shorten the detection time from 3-5 days of the traditional method to 3 hours, and achieve the following detection effects:
Multiple detection coverage
The detection method established by the invention can check whether the Escherichia coli and the Shigella are contained in one PCR reaction and determine the pathotypes of the six Escherichia coli. The detection result can be rapidly obtained within 3 hours, and the time, labor and material cost are saved.
(II) high specificity
The specificity of the detection method established by the invention is mainly reflected in the specificity of a whole set of primers, and all the primers are subjected to blast comparison analysis and have high conservatism and specificity; meanwhile, a specificity experiment shows that the primer can well distinguish bacteria which have similar species and same living environment with the Escherichia coli and Shigella diarrhoea, including Enterobacter sakazakii, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Listeria monocytogenes, Vibrio cholerae, Bacillus cereus, Campylobacter jejuni, Campylobacter coli, Enterobacter sakazakii, Yersinia enterocolitica, Aeromonas hydrophila and the like.
(III) high sensitivity
The detection method established by the invention can realize the simultaneous detection of 14 genes, and the detection sensitivity of each target gene in a reaction system can reach 103CFU/ml, which is equivalent to the detection sensitivity of single real-time fluorescent PCR; meanwhile, the detection sensitivity of different genes is on the same horizontal line, so that the omission of mixed infection of six kinds of diarrhea escherichia coli and shigella is avoided. The main reason for improving the sensitivity of the invention is that the invention designs a specific LHP sequence aiming at the specific primer pair sequences of all target genes, the homologous universal primer in the LHP sequence can improve the amplification efficiency of multiple PCR reaction in the second stage PCR, reduce the non-specific amplification of the multiple PCR reaction and the generation of primer dimer, and the 6 basic group connecting sequence in the LHP can ensure that the homologous universal primer has the same amplification efficiency when amplifying different virulence genes, avoid the unbalance of the yield of different target genes in the multiple PCR amplification process, thereby improving the sensitivity of the multiple PCR detection on the whole.
(IV) the cost is lower
The multiple PCR detection method established by the invention reduces the labor cost and time cost on the operability, the original single detection needs 14 times of labor and 14 times of time, and only 1 time of labor and 1 reaction time is needed by using the method; the multiple detection method simultaneously saves the reagent consumption for repeatedly detecting the same sample, and can maximally save more than 50% of the reagent cost.
(V) prevention of false negative results
The positive internal control added in the system can effectively prompt false negative detection results.
The invention provides a complete solution for rapidly screening six kinds of Escherichia coli and Shigella dysenteriae, can realize rapid identification of six kinds of Escherichia coli pathotypes and Shigella dysenteriae after occurrence of food poisoning events and outbreak of epidemic disease, and provides reliable basis for reasonably and properly treating epidemic disease.
The invention overcomes the defects of complex operation, time and labor consumption, difficulty in uniform quality control and the like in the prior art, and solves the problem of simultaneous and rapid identification of six kinds of diarrhea escherichia coli and shigella.
Based on an IpaH gene of EIEC/Shigella, a universal gene uidA of Escherichia coli/Shigella and related virulence genes thereof, a set of six primer sequences for identifying Escherichia coli and Shigella are designed, a multiplex PCR detection method based on the primer sequences is established, an amplification internal standard is added into the system, and internal quality control is completed in one reaction system.
additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The invention provides a primer group for detecting six kinds of diarrhea escherichia coli and shigella by multiplex PCR, wherein the primer group comprises primers shown in SEQ ID NO. 1-29; the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible-adhering Escherichia coli.
Wherein, the nucleotide sequence of the homologous universal primer for detecting six kinds of diarrhea Escherichia coli and Shigella is shown as SEQ ID NO. 29;
The invention also provides an LHP sequence for detecting the long primers of six kinds of diarrhea Escherichia coli and Shigella, and the nucleotide sequence is shown as SEQ ID NO. 30;
Wherein the primer group also comprises primers shown in SEQ ID NO. 31-32. The primers shown in SEQ ID NO.31-32 are a pair of primers designed by using pET28a plasmid as a template and used as a positive internal control primer pair.
The primer group provided by the invention is designed by taking 14 genes as target amplification sequences, wherein the uidA gene is a molecular target for judging Escherichia coli and Shigella; elt, estla and estlb are ETEC identification targets; bfpB and escV are identification targets of EPEC; escV, stx1 and stx2 are identification targets of EHEC; ipaH is an identification target of EIEC; the astA, aggR and pic are identification targets of the EAEC; daaC and afa were identified targets of DAEC (as shown in table 1).
TABLE 1 summary of six kinds of primers for identifying Escherichia coli and Shigella diarrhoea by multiplex PCR
In one embodiment of the present invention, there is provided a method for detecting six kinds of Escherichia coli and Shigella dysenteriae, comprising the steps of: (1) extracting the total DNA of a sample to be detected; (2) carrying out multiple PCR reaction by using the total DNA as a template and the primer group to obtain a material subjected to multiple PCR amplification; (3) and carrying out nucleic acid electrophoresis detection on the material subjected to the multiple PCR amplification to obtain a nucleic acid electrophoresis detection result. And judging the sample according to the result of nucleic acid electrophoresis. The sample judgment criteria are shown in table 2.
TABLE 2 sample criteria
Among them, it is preferable that the primers shown by SEQ ID NO.1 to 28 are used at final concentrations of 0.1 to 0.8. mu.M, respectively, and the primer shown by SEQ ID NO.29 is used at final concentrations of 0.4 to 0.8. mu.M.
wherein, preferably, the conditions of the multiplex PCR reaction comprise the following steps a-h:
a：94-96℃，4-6min；
b：94-96℃，15-60s，
c：50-60℃，15-60s，
d: carrying out 10-15 b-d cycles at the temperature of 71-73 ℃ for 60-120 s;
e：94-96℃，15-60s，
f：55-65℃，15-60s，
g: 30-35 e-g cycles are carried out at the temperature of 71-73 ℃ for 30-120 s;
h：71-73℃，5-10min。
Wherein, the sample to be tested can include but is not limited to at least one of food, medicine, excrement, vomit and body fluid. Preferably, the detection method for detecting six kinds of Escherichia coli and Shigella diarrheagenic bacteria by multiplex PCR of the present invention is not used for diagnosis. Or the qualitative and quantitative results of the six kinds of Escherichia coli and Shigella dysenteriae have no one-to-one correspondence with whether the disease occurs, which does not belong to the diagnosis result, but can be used as intermediate information for reference of a clinician.
Wherein, preferably, the nucleic acid electrophoresis detection comprises nucleic acid gel electrophoresis and/or nucleic acid capillary electrophoresis.
In one embodiment of the present invention, a QIaxcel fully automated capillary electrophoresis analyzer was used as a platform for the analysis of results. And based on the full-automatic operation of QIaxcel and a set intelligent result analysis program, the automatic judgment and analysis of the result are realized.
One aspect of the invention also provides a kit for detecting six kinds of Escherichia coli and Shigella diarrhoea by multiplex PCR, wherein the kit comprises the primer set and the DNA polymerase; the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible-adhering Escherichia coli.
Preferably, the kit further comprises PCR reaction reagents required for PCR reaction, and the reaction reagents can be PCR reaction buffer and dNTP.
On the other hand, the invention also provides the application of the primer group in the preparation of a kit for detecting six kinds of Escherichia coli and Shigella dysenteriae; wherein the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible Escherichia coli.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the following examples, both enteroinvasive Escherichia coli and Shigella are from the China center for medical bacterial Collection.
EXAMPLE 1 construction of six types of multiple PCR detection kits for the identification of Escherichia coli and Shigella dysenteriae
The kit consists of 2 multiplied reaction system buffer solution, DNA polymerase, 10 multiplied primer mixed solution, positive control and deionized water, and comprises the following specific components: 2 XPCR Buffer (Tris HCl 40mM (pH8.3), KCl 100mM, tween-200.08%, 0.0006 ng/. mu.L pET28a, 1.5mM dNTP, 8mM MgCl 2); 25XDNA polymerase (2U/. mu.L); 10 × primer mixture (including IAC primer concentration of each long primer 2 μ M, homologous universal primer SEQ ID NO.29 concentration of 8 μ M), positive control (EPEC, EHEC, ETEC, EAEC, DAEC, EIEC/Shigella mixed template, each 106CFU/mL), positive internal control pET28a plasmid.
The reaction system for detecting the kit is 25 mu L, and the configuration is as follows: 2 × PCR Buffer 12.5 μ L; 25 × DNA polymerase 1 μ L; 10 × 2.5 μ L of primer mixture; template 2. mu.L, deionized water 7. mu.L.
Example 2 operation and result judgment of the kit
1. Extraction of nucleic acids
And (3) dissolving enrichment fluid or colony cultured by streaking in water, and extracting template DNA according to the instruction of the bacterial genome extraction kit. The concentration of total extracted DNA was determined using NanoDrop.
2. preparation of the reaction System
A25. mu.L reaction system was prepared in a 200. mu.L PCR tube as follows: 2 × PCR Buffer 12.5 μ L; 25xDNA polymerase 1. mu.L; 10 times the primer mixture 2.5. mu.L, template 2. mu.L, deionized water 7. mu.L.
3. PCR reaction
The PCR tube was placed in a Bio-Rad C1000 PCR apparatus, and after opening the hot lid, PCR was performed according to the following procedure:
a：95℃4min；
b：95℃30s，
c：55℃30s，
d: 90s at 72 ℃, and 10 reactions are circulated in the b-d way;
e：95℃30s，
f：60℃30s，
g: circulating 30 reactions at 72 ℃ for 90s and e-g;
h：72℃，5min。
4. Full-automatic capillary electrophoresis analysis of PCR products
The PCR reaction tube is placed into a full-automatic capillary electrophoresis apparatus Qiaxcel Advanced (Kajie corporation), a high-resolution clamp is used, a proper alignment marker and a proper size marker are selected, and the size of a target strip is automatically analyzed.
5. Result judgment
A. All lanes including the blank have at least one amplified band, the blank has the amplified positive inner control, and the other detected target bands and/or amplified positive inner control show that all PCR reactions are established and false negative results are excluded. Otherwise, the experiment is invalid and needs to be repeated.
B. All samples had amplified bands of uidA (1673bp), i.e.Escherichia coli or Shigella. Other samples also amplified virulence genes, which are diarrheagenic E.coli. On this basis, the specific determination is as follows:
The sample has amplification bands of uidA (1673bp) and escV (548bp), and also has amplification band of bfpB (172bp), and the sample is judged as typical EPEC because the two shiga toxins stx1(242bp) and stx2(305bp) are negative.
When the sample has uidA (1673bp) and escV (548bp) band amplification, two shiga toxins stx1(242bp) and stx2(305bp) are positive, and bfpB (172bp) is not amplified, the sample is judged to be EHEC.
When the sample has uidA (1673bp) band amplification, the heat-labile enterotoxin elt (339bp) and the heat-labile enterotoxin estIb (221bp) are positive, and the sample is judged as ETEC.
When the sample has uidA (1673bp) band amplification, ipaH (433bp) is positive, and the EIEC/Shigella is judged.
When the sample has uidA (1673bp) band amplification, the astA (153bp), the aggR (492bp) and the pic (965bp) are all positive, and the sample is judged to be EAEC.
while the samples were amplified with uidA (1673bp) and escV (548bp) bands, stx1(242bp), stx2(305bp) and bfpB (172bp) were all negative, and thus the samples were judged as atypical EPEC.
When the sample has uidA (1673bp) band amplification, the heat-labile enterotoxin elt (339bp) and the astA (153bp) are positive, and under the condition of existing with other type virulence genes at the same time, the astA is not used as a judgment basis, and the sample is judged as ETEC.
When the sample has uidA (1673bp) band amplification, the astA (153bp) is positive, and the sample is judged as EAEC.
All target bands contained in the positive control can be amplified, and the kit has the capability of detecting mixed infection.
EXAMPLE 3 specificity test of the kit
Enterobacter sakazakii (purchased from China medical strain collection center, number 21665), Staphylococcus aureus (purchased from China medical strain collection center, number 26003), Salmonella (purchased from China medical strain collection center, number 50001), Vibrio parahaemolyticus (purchased from China medical strain collection center, number 21617), Listeria monocytogenes (purchased from China medical strain collection center, number 54002), Vibrio cholerae (purchased from China medical strain collection center, number 54002), campylobacter jejuni (purchased from China medical strain collection center, number 54102), campylobacter coli (purchased from China medical strain collection center, number 47016), Aeromonas hydrophila (purchased from China medical strain collection center, number 44016) were selected as bacteria to be tested. When the kit is used for detecting the bacteria to be detected, amplification internal standards are positive, and the establishment of a detection system is proved; no non-specific miscellaneous band appears in the bacteria to be detected. And no impurity band is generated except a specific band when the target bacteria are detected. The kit can effectively distinguish non-target bacteria and has better specificity.
Example 4 minimum detection Limit test of the kit
test samples for evaluation: selection of the particular strains tested: EPEC, EHEC, ETEC, EAEC, DAEC, EIEC/Shigella. The concentrations of the 6 templates are respectively adjusted to 108CFU/mL, and the templates are mixed into a comprehensive template in equal proportion. The integrated template gradient was diluted to 106CFU/mL, 105CFU/mL, 104CFU/mL, 103CFU/mL, 102CFU/mL of the test sample.
The kit of the invention is used for detecting mixed templates with different dilutions respectively. The results of the lowest detection limit test for 14 target genes (without IAC) with the kit according to the procedure and result judgment of example 2 are shown in Table 3:
Table 3 test results of minimum detection limit of 14 target genes detected by kit
| Numbering | Amplifying band names | 106CFU/mL | 105CFU/mL | 104CFU/mL | 103CFU/mL | 102CFU/mL |
| 1 | ipaH | + | + | + | + | - |
| 2 | uidA | + | + | + | + | - |
| 3 | elt | + | + | + | + | - |
| 4 | estIa | + | + | + | + | - |
| 5 | estIb | + | + | + | + | - |
| 6 | bfpB | + | + | + | + | - |
| 7 | escV | + | + | + | + | - |
| 8 | stx1 | + | + | + | + | - |
| 9 | stx2 | + | + | + | + | - |
| 10 | astA | + | + | + | + | - |
| 11 | aggR | + | + | + | + | - |
| 12 | pic | + | + | + | + | - |
| 13 | afa | + | + | + | + | - |
| 14 | daaC | + | + | + | + | - |
As shown in Table 3, the kit can detect six kinds of Escherichia coli and Shigella at the lowest detection limit of 103 CFU/mL.
example 5 comparison of kit assays with literature methods
The kits of the present invention were evaluated in comparison with the results reported in the literature (Claudia Toma, Yan Lu,1 Naomi Higa, Noboru Nakasone, Isabel Chinen, Ariela Baschkier, Marta Rivas, and Masaaki Iwana. multiplex PCR Assay for Identification of Human diarrheeal Escherichia coli. American Society for Microbiology, dec.2004, p.5849-5853), and were compared mainly in terms of the minimum detection limit, specificity and coverage.
Positive sample for evaluation: EPEC, EHEC, ETEC, EAEC, DAEC, EIEC, and Shigella each 1 strain, for a total of 7 strains.
Negative samples for evaluation: enterobacter sakazakii (purchased from china medical strain collection center, number 21665), staphylococcus aureus (purchased from china medical strain collection center, number 26003), salmonella (purchased from china medical strain collection center, number 50001), vibrio parahaemolyticus (purchased from china medical strain collection center, number 21617), listeria monocytogenes (purchased from china medical strain collection center, number 54002), vibrio cholerae (purchased from china medical strain collection center, number 54002), campylobacter jejuni (purchased from china medical strain collection center, number 54102), campylobacter coli (purchased from china medical strain collection center, number 47016), aeromonas hydrophila (purchased from china medical strain collection center, number 44016). After nucleic acid is extracted from the strains, the strains are mixed in equal proportion to form a negative evaluation DNA template.
And (3) specific evaluation: the specificity evaluation procedure was carried out according to examples 1 and 2 of the present invention; uidA, ipaH, elt, estIa, estIb, bfpB, escV, stx1, stx2, astA, aggR, pic, afa, daaC genes and positive internal controls were tested according to literature reports.
Coverage evaluation: the 7 positive samples for evaluation are respectively detected by adopting the kit and a literature method, and the DNA template of each strain is 5-10 ng/uL.
Evaluation of minimum detection limit: diluting all 7 positive samples for evaluation into 106CFU/mL, 105CFU/mL, 104CFU/mL, 103CFU/mL and 102CFU/mL respectively, extracting nucleic acid to prepare a positive sample DNA template, and detecting the gradient diluted DNA templates of the 7 positive samples for evaluation by adopting the kit and a literature method.
And (3) comparing the results: the results of the kit and the literature report method for detecting the negative samples are negative, which shows that the kit and the literature report method have better specificity.
The kit and the literature report method detect target genes in 7 positive samples for evaluation, and positive results are obtained, wherein the literature report method can only detect ipaH, eae, elt, est, aggR and stx genes; the kit can detect all kinds of target genes in a positive sample, including uidA, ipaH, elt, estIa, estIb, bfpB, escV, stx1, stx2, astA, aggR, pic, afa and daaC genes and positive internal control.
the lowest detection limit of the kit and the literature report method for detecting the target genes in 7 positive samples is shown in the following table 4:
TABLE 4
As can be seen from the above table, the kit of the present invention can detect uidA, ipaH, elt, estIa, estIb, bfpB, escV, stx1, stx2, astA, aggR, pic, afa and daaC target genes with minimum detection limits reaching 103CFU/mL, whereas the literature reports that only ipaH can reach 103CFU/mL, eae, elt, est and stx can reach 104CFU/mL, and aggR can reach 105 CFU/mL. The lowest detection limit of the kit is obviously superior to that of a method reported in the literature.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (9)
1. A primer group for detecting six kinds of Escherichia coli and Shigella diarrhoea by multiplex PCR, wherein the primer group comprises primers shown as SEQ ID NO. 1-29; the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible-adhering Escherichia coli.
2. The primer set according to claim 1, wherein the primer set further comprises primers represented by SEQ ID nos. 31 to 32.
3. A method for detecting six species of escherichia coli and shigella dysenteriae for non-diagnostic purposes, comprising the steps of:
(1) extracting the total DNA of a sample to be detected;
(2) Performing a multiplex PCR reaction using the total DNA as a template and the primer set of claim 1 or 2 to obtain a material after multiplex PCR amplification;
(3) and carrying out nucleic acid electrophoresis detection on the material subjected to the multiple PCR amplification to obtain a nucleic acid electrophoresis detection result.
4. The detection method according to claim 3, wherein the primers shown by SEQ ID NO.1 to 28 are used at a final concentration of 0.1 to 0.8. mu.M, respectively, and the primers shown by SEQ ID NO.29 are used at a final concentration of 0.4 to 0.8. mu.M.
5. The detection method according to claim 3 or 4, wherein the conditions of the multiplex PCR reaction include the following steps a-h:
a：94-96℃，4-6min；
b：94-96℃，15-60s，
c：50-60℃，15-60s，
d: carrying out 10-15 b-d cycles at the temperature of 71-73 ℃ for 60-120 s;
e：94-96℃，15-60s，
f：55-65℃，15-60s，
g: 30-35 e-g cycles are carried out at the temperature of 71-73 ℃ for 30-120 s;
h：71-73℃，5-10min。
6. The detection method according to claim 3 or 4, wherein the nucleic acid electrophoresis detection comprises nucleic acid gel electrophoresis and/or nucleic acid capillary electrophoresis.
7. A kit for detecting six kinds of Escherichia coli and Shigella diarrheal by multiplex PCR, which comprises the primer set of claim 1 or 2 and DNA polymerase; the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible-adhering Escherichia coli.
8. The kit of claim 7, wherein the kit further comprises PCR reaction buffer and dNTPs.
9. Use of the primer set of claim 1 or 2 for the preparation of a kit for detecting six species of Escherichia coli and Shigella dysenteriae; wherein the six diarrheagenic Escherichia coli bacteria include enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, and enterodiffusible Escherichia coli.
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| CN109576385B (en) * | 2018-12-18 | 2019-12-20 | 北京卓诚惠生生物科技股份有限公司 | Nucleic acid reagent, kit and system for detecting escherichia coli |
| CN111518934A (en) * | 2020-05-21 | 2020-08-11 | 四川大学 | Rapid detection method and kit for escherichia coli and 6 pathogenic genes |
| CN112481400A (en) * | 2020-12-24 | 2021-03-12 | 山西大学 | Rapid detection kit and method for food pathogenic bacteria diarrhea causing escherichia coli |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451305A (en) * | 2013-09-17 | 2013-12-18 | 北京卓诚惠生生物科技有限公司 | Primers, probe, method and kit for detecting diffusely adherent Escherichia coli |
| CN103484546A (en) * | 2013-09-17 | 2014-01-01 | 北京卓诚惠生生物科技有限公司 | Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit |
| CN103451306B (en) * | 2013-09-17 | 2014-11-19 | 北京卓诚惠生生物科技有限公司 | Multi-PCR (Polymerase Chain Reaction) detecting primer group for Escherichia coli O104:H4 and reagent kit |
-
2016
- 2016-08-31 CN CN201610799754.7A patent/CN106434901B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451305A (en) * | 2013-09-17 | 2013-12-18 | 北京卓诚惠生生物科技有限公司 | Primers, probe, method and kit for detecting diffusely adherent Escherichia coli |
| CN103484546A (en) * | 2013-09-17 | 2014-01-01 | 北京卓诚惠生生物科技有限公司 | Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit |
| CN103451306B (en) * | 2013-09-17 | 2014-11-19 | 北京卓诚惠生生物科技有限公司 | Multi-PCR (Polymerase Chain Reaction) detecting primer group for Escherichia coli O104:H4 and reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| 3种致泻性大肠埃希氏菌多重PCR检测方法的研究;张铁男等;《中国兽医杂志》;20071231;第43卷(第9期);17-18 * |
| multiplex pcr assay for identification of human diarrheagenic escherichia coli;claudia toma et al.;《journal of clinical microbiology》;20030630;2669-2671 * |
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