CN107119131B

CN107119131B - Kit for simultaneously detecting serotype of Escherichia coli O1-O7

Info

Publication number: CN107119131B
Application number: CN201710349453.9A
Authority: CN
Inventors: 李群; 郑翰; 王红; 熊衍文; 李新琼; 张正东; 刘祥; 张玲; 邹年莉; 闫国栋
Original assignee: Zigong Municipal Center For Disease Control And Prevention
Current assignee: Zigong Municipal Center For Disease Control And Prevention
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2020-03-17
Anticipated expiration: 2037-05-17
Also published as: CN107119131A

Abstract

The invention provides a kit for simultaneously detecting Escherichia coli O1-O7 serotypes, and belongs to the technical field of multiplex PCR detection. The kit comprises a specific primer pair and a pair of internal reference primers aiming at serotypes O1-O7 of Escherichia coli respectively, wherein the nucleotide sequences of the specific primer pair are respectively shown as SEQ ID NO.1-14, and the nucleotide sequences of the internal reference primers are respectively shown as SEQ ID NO. 15-16. The kit provided by the invention realizes accurate detection of the serotype of Escherichia coli O1-O7 by setting a multiple PCR system, optimizing reaction conditions, analyzing the product length through single-tube one-time PCR and agarose electrophoresis. The kit has the advantages of accurate detection, high sensitivity, strong specificity, simplicity, convenience and rapidness, is suitable for epidemiological investigation of the Escherichia coli in a clinical diarrhea sample, and has good application prospect.

Description

Kit for simultaneously detecting serotype of Escherichia coli O1-O7

Technical Field

The invention relates to the technical field of multiplex PCR detection, in particular to a method for simultaneously detecting Escherichia coli O1-O7 serotypes, a detection kit and application thereof.

Background

Escherichia coli (e.albertii) is a new enteropathogenic bacterium discovered and named in recent years that is associated with the outbreak of intestinal infections and food-borne diarrhea in humans. In 1991, Albert et al isolated Escherichia Albert for the first time from stool specimens of children with diarrhea in Bengal Lacca with vomiting, fever, mild dehydration and abdominal distension. In 2003, Huys et al identified it as a new species in the genus Escherichia by phenotypic and genotypic analyses and was named E.albertii (Escherichia albertii). In 11 months 2011, in autumn county of japan, an outbreak of an escherichia coli-associated gastroenteritis case occurred. In addition, the strain was isolated from diarrhea patients, Guinea Pisha healthy people, in Minnesota and Illinois, USA. 1993 Ooka et al isolated 13 strains of Escherichia coli from patients with gastrointestinal infections in Japan, Belgium, Brazil, Germany, and 1 strain from asymptomatic carriers. It is speculated that the bacterium Albert may be one of the causes of 62,000,000 and 3,200 deaths of food-borne diseases each year in the United states due to unknown causes. In addition, foreign scholars have also isolated Escherichia coli from animal hosts such as domestic fowls, domestic animals, and birds. The applicant firstly proves the existence of the Escherichia coli in Chinese diarrhea patients, healthy people, wild aigret, poultry and animal-derived foods, and obtains 62 strains of Escherichia coli isolates from different sources in China, wherein MLST and PFGE types of the strains have obvious diversity. The monitoring research result indicates that the Escherichia coli has a larger risk of human-to-human infection in China.

Lipopolysaccharide (LPS) is gram negative (G)^-) The important components of the bacterial outer membrane play an important role in the survival of bacteria in the environment and hosts. The O-antigen polysaccharide is not only an important component of LPS, but also G^-The determining component of the bacterial serodiversity. There are significant differences in the composition and structure of O-antigens in the same species of bacteria. Serotyping techniques based on the recognition of different kinds of O antigens in LPS would provide very valuable information in the work of monitoring of Escherichia coli and epidemiological studies. There has not been any report on the study of the Escherichia coli O antigen and its serotyping. The chemical composition and structure of the O antigen also vary significantly among bacteria of the same species, making it the basis for serological diversity. Serotypes remain the "gold standard" for monitoring and identifying bacteria, have important clinical and epidemiological significance, and have important instructional significance for vaccine development.

In previous studies, seven serotypes of Escherichia coli were found, named O1-O7, and diagnostic sera corresponding to each serotype were successfully prepared, all of which specifically agglutinated only with homotypic strains and did not cross-agglutinate with non-homotypic strains. However, conventional serotyping techniques use diagnostic serum for agglutination tests, which is time consuming, laborious and expensive. The molecular serotyping technology based on the detection of different serotype specific genes has the characteristics of high flux, rapidness, simplicity, convenience, specificity and economy, and gradually replaces the traditional seroagglutination method. In order to further understand the serotype epidemiological information of Escherichia coli and understand the dominant serotype of Escherichia coli existing in China, a high-throughput molecular typing technique for detecting the serotype needs to be developed.

Disclosure of Invention

The invention aims to provide a multiplex PCR method for simultaneously detecting Escherichia coli O1-O7 serotypes; the invention also aims to provide a kit for simultaneously detecting the serotype O1-O7 of the Escherichia coli and application thereof, so that the identification process of the serotype O1-07 of the Escherichia coli is simplified, and the detection rate of pathogenic bacteria is improved to further know the serotype epidemiological information of the Escherichia coli.

The O-antigen synthesis related gene is located on the chromosome of the bacteria and is called O-antigen biosynthesis related gene cluster (O-AGC). The invention determines the available target genes and develops a multiplex PCR detection method capable of simultaneously detecting 7 types of O-AGCs by analyzing the characteristics of the O-AGCs in the Escherichia coli strain. Applicants have discovered that the isolated 62 strains of Escherichia albertiae possess an intact O-AGC as a result of their O-AGCs sequencing. Similar to many enteropathogens, all O-AGCs of Escherichia albertiae are located in a fixed region of the genome between the galF and gnd genes. The possession of wzy/wzx genes in each O-AGC suggests that the O antigen of Escherichia albertiae is also synthesized via the Wzx/Wzy pathway, and that wzy genes have high intra-type conservation. Aiming at the gene, the invention designs a specific primer, adds an Escherichia coli species specific gene detection primer, establishes an O1-O7 serotype multiplex PCR detection method by optimizing the primer concentration and a reaction program, can identify 7 types of O-AGCs simultaneously in a single reaction, and can also determine whether the detected strain is Escherichia coli.

The invention firstly provides a specific primer pair combination for simultaneously detecting serotype O1-O7 of Escherichia coli, which comprises the following primer pairs:

the nucleotide sequence of the specific primer pair for detecting the serotype O1 of the Escherichia coli is shown as SEQ ID NO. 1-2;

the nucleotide sequence of the specific primer pair for detecting the serotype O2 of the Escherichia coli is shown as SEQ ID NO. 3-4;

the nucleotide sequence of the specific primer pair for detecting the serotype O3 of the Escherichia coli is shown as SEQ ID NO. 5-6;

the nucleotide sequence of the specific primer pair for detecting the serotype O4 of the Escherichia coli is shown as SEQ ID NO. 7-8;

the nucleotide sequence of the specific primer pair for detecting the serotype O5 of the Escherichia coli is shown as SEQ ID NO. 9-10;

the nucleotide sequence of a specific primer pair for detecting the serotype O6 of the Escherichia coli is shown as SEQ ID NO. 11-12;

the nucleotide sequence of the specific primer pair for detecting the serotype O7 of the Escherichia coli is shown as SEQ ID NO. 13-14.

The invention provides application of the specific primer pair combination in preparing a kit for simultaneously detecting the serotype O1-O7 of Escherichia coli.

The invention provides an application of an internal reference primer and the specific primer pair in combination and use in preparation of a kit for detecting Escherichia coli serotype O1-O7, wherein the nucleotide sequence of the internal reference primer is shown as SEQ ID NO. 15-16.

Further, the invention provides a detection kit, which comprises the specific primer pair combination and a pair of internal reference primers, wherein the nucleotide sequence of the internal reference primers is shown as SEQ ID NO.15-16, and the specific primer pair combination comprises the primer sequence shown as SEQ ID NO. 1-14.

The working procedure of the kit comprises the following steps:

(1) extracting nucleic acid of a sample to be detected;

(2) carrying out single-tube once multiplex PCR reaction on the DNA of a sample by using the specific primer pair combination and the internal reference primers shown in SEQ ID NO. 15-16; detecting the PCR product by electrophoresis, judging whether the sample contains the Escherichia coli and which one of serotypes O1-O7 according to the fragment size of the PCR product,

if the PCR product fragment is 554bp, the serotype of Escherichia coli O1 exists in the sample to be detected;

if the PCR product fragment is 304bp, the serotype of Escherichia coli O2 exists in the sample to be detected;

if the PCR product fragment is 173bp, the serotype of Escherichia coli O3 exists in the sample to be detected;

if the PCR product fragment is 139bp, the serotype of Escherichia coli O4 exists in the sample to be detected;

if the PCR product fragment is 287bp, the serotype of Escherichia coli O5 exists in the sample to be detected;

if the PCR product fragment is 413bp, the serotype of Escherichia coli O6 exists in the sample to be detected;

if the PCR product fragment is 758bp, the Escherichia coli O7 serotype exists in the sample to be detected.

In the multiplex PCR reaction, the concentration of each primer in the PCR reaction solution is respectively as follows:

wherein the multiple PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min;

30s at 94 ℃, 30s at 64 ℃, 60s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.

The invention provides application of the kit in non-diagnostic detection of Escherichia coli O1-O7 serotype.

For the analysis of the amplification products, the present invention can use any high resolution (band size difference can be distinguished by more than 5 bp) electrophoresis technique. The multiplex PCR results of the kit of the present invention can be analyzed by using a capillary electrophoresis apparatus with High Resolution, for example, a DNA High Resolution kit for fragment analysis on a QIAxcel apparatus, and DNAalignment Marker 15bp/600bp and size Marker 25bp/500bp for determining the size of the fragment.

The invention designs specific primers for detecting different serotypes aiming at wzy genes of the serotypes O1-O7 of the Escherichia coli, avoids the primers from forming a secondary structure, and avoids the occurrence of mismatching and nonspecific fragments; in the multiplex PCR system, when designing the primer sequences, the TM values of the upstream and downstream primers are considered to be as close as possible, the size of each amplification product is between 100bp and 500bp, and the difference between the sizes is more than 5 bp.

According to the invention, by setting a multiple PCR system and optimizing the configuration of the reaction system, the temperature, the time and the like of the PCR reaction, the accurate detection of the serotype of the Escherichia coli O1-O7 is realized. The invention can simultaneously detect 7 targets in a single PCR amplification system, covers serotypes O1-O7 of the Escherichia coli, enables identification of serotypes O1-O7 of the Escherichia coli to be simpler and more broad-spectrum, and can simultaneously detect 7 serotypes and determine whether a detected strain is the Escherichia coli or not in one PCR reaction. The kit has the advantages of accurate detection, high sensitivity, strong specificity, simplicity, convenience and rapidness, is suitable for epidemiological investigation of the Escherichia coli in a clinical diarrhea sample, and has good application prospect.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of serotype E.albertzeri O1-O7 detected by the kit of the present invention.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; all reagents used in the examples are commercially available unless otherwise specified.

EXAMPLE 1 design of specific primers

1. The strain source is an Albert Hirschmanni reference strain LMG20976, a feces isolate of a Shanghai diarrheal patient in 2013, 62 strains isolated from multiple sources in 2014-2016 of tribute City, Sichuan province, and 64 strains in total.

2. Whole genome sequencing and bioinformatics analysis

Whole genome DNA was extracted and purified using Wizard Genomic DNA Purification kit (Promega, Madison, USA). After sequencing the genome of the strain by Illumina Solexa GA IIx (Illumina, San Diego, Calif.), a paired-end (PE) library with an average insertion length of 500 bp-2000 bp is constructed, and data are assembled by SOAP denovo (Release 1.04). Open Reading Frames (ORFs) were identified and annotated by the Aremis program (www.sanger.ac.uk), BLAST and PSI-BLAST software (http:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi) was used to search GenBank (www.ncbi.nlm.nih.gov/GenBank), (COG; www.ncbi.nlm.nih.gov/COG), and Pfam (Pfam. sanger. ac. uk) protein databases to extract each O-AGC between galF and gnd genes from genomic sequences. TMHMM v2.0(http:// www.cbs.dtu.dk/services/TMHMM /) was used to analyze the hydrophobic transmembrane domains of proteins.

All O-AGCs of Escherichia albertiae are located in the region between galF (encoding UTP-glucose-1-phosphate uridyltransferase) and gnd (6-phosphogluconate dehydrogenase). All O-AGCs carry wzx and wzy genes. The DNA sequence homology of wzy and wzx genes is more than 99.9 percent between the same serotype strain, and the DNA sequence homology of wzy and wzx genes between different serotype strains is less than 8 percent. The 7O-AGCs ranged in size from 7.2kb (O5, including 7 genes) to 16.4kb (O4, including 16 genes), with the G + C content of the 7O-AGCs ranging from 29.16% (O5) to 38.82% (O4). The result shows that the O antigen of the Escherichia coli is synthesized through Wzx/Wzy, and wzy gene plays a key role in O-antigen synthesis, the sequence is conserved in types, and the difference between types is great, so the gene is an ideal target gene for detection.

3. Design of specific primers were designed against specific wzy sequences of the type of Escherichia coli O-AGCs, and in order to enable the primers to work in multiplex PCR experiments under the same conditions and system, Primer-BLAST program (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) was used to design primers with essentially similar parameters for each pair of primers, as listed in Table 1, while the E.coli housekeeping gene (Eal-O) from published literature was used as an internal control.

TABLE 1 specific primer sequences of the invention and related parameters

In the invention, the primers are key factors of multiplex PCR, and when the primers are designed, (1) the primers with higher specificity should be selected from nucleotide fragments with high conservation, but when the primers are designed, not only are highly conserved regions of wzy genes among all serotypes considered, but also specific fragments which simultaneously represent all serotypes in the conserved regions are required to be ensured, and the specificity among all the highly conserved regions is also required to be ensured, so that different serotypes are easy to distinguish.

(2) Secondary structures are prevented from forming among the primers, such as a self hairpin structure, an upstream primer and a downstream primer, or a dimer is formed between the primers and the probes, and the primers are prevented from being mismatched with the target gene, so that non-specific fragments can be generated if one primer is combined with multiple places of the target gene.

(3) The 3 'end of the primer must remain highly conserved, and during annealing, the 3' end of the primer is where extension begins, so that replication can be performed with maximum efficiency.

(4) The Tm value is an important determinant in designing primers, and provides reference data for optimal annealing temperature of primers, and the Tm values of upstream and downstream primers should be close to each other as possible, and at the same time, the Tm values of all upstream and downstream primers should not exceed 10 ℃ at most, considering that 12 diarrhea pathogens are prepared for multiplex PCR.

(5) Because the serotypes of the Escherichia coli are detected simultaneously, the cross reactions among 7 pairs of primers, between a primer pair and a template, between an internal reference primer and 7 pairs of specific primers and between the internal reference primer and different PCR products are avoided, and finally the optimal primer pair combination in the table 1 is screened out.

EXAMPLE 2 establishment of multiplex PCR detection System for simultaneously detecting serotype of Escherichia coli O1-O7

1. Optimization of multiplex PCR systems

By adjusting the concentration of each pair of primers, the final amplification system is determined to be 20 μ l: o1 and O2 upstream and downstream primers, 0.15. mu.l each; o7 upstream and downstream primers, 0.25. mu.l each; 0.5 mul of each of the upstream and downstream primers of O4 and O6; o3 upstream and downstream primers, 0.2. mu.l each; o5 upstream and downstream primers, 0.3. mu.l each; Eal-O upstream and downstream primers each at 1.5. mu.l (wherein the concentrations of the 7 pairs of primers except O6 primer were 10. mu. mol/L, respectively); 1. mu.l of DNA; double distilled water 1.9 ul; 1ul of template; 2 XTaq PCR MasterMix (supplied by Beijing Bomaide) 10. mu.l.

2. The thermal cycle parameters are optimized.

According to the TM value of the primer, 6 gradients of 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃ and 64 ℃ are selected for parameter optimization, the amplification effect of 8 pairs of primers is integrated, and 64 ℃ is selected as the final annealing temperature.

3. Establishment of multiplex PCR System

Using the primers shown in Table 1 of example 1, the specificity of each pair of primers was first tested by performing a single PCR amplification using the genomic DNA of the reference strains of the 7 serotypes of Escherichia coli of example 1 as a template and double distilled water without nucleic acid as a negative control. As a result, in the single PCR, the serotype corresponding to each pair of primers has 100% specificity, and a system for detecting 7 types of serotypes of Escherichia coli by a multiplex PCR method is further established.

The DNA of 7 reference strains is used as a template to test each group of systems, the results of gel electrophoresis analysis show that all types have positive bands aiming at the 846bp fragment of the internal control gene Eal-o, but only the corresponding types can have the positive bands aiming at the wzy gene, and other types do not have non-specific amplification bands, and the wzy positive bands of the same group can be well distinguished according to different fragment sizes (figure 1). The size of the amplified band of each primer is shown in Table 1. The detection result is in accordance with the expected result.

The condition is pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 64 ℃, 60s at 72 ℃ and 35 cycles; finally, the extension is carried out at 72 ℃ for 5 min. After the amplification is finished, agarose gel electrophoresis is carried out to detect a reaction band. The size of the band of interest for each serotype is shown in table 1.

4. Determination of detection result

Different serotypes amplified the expected specific PCR products and presented good separation on a 1.5% agarose gel, with each PCR product size clearly distinguishable as shown in figure 1. The expected product sizes for the reference strains were as follows: o1(554bp), O2(304bp), O3(173bp), O4(139bp) O5(287bp), O6(413bp) and O7(758 bp). The lower detection limit of the detection system is 10-50 pg. The results of the test on 64 strains of Escherichia coli were completely consistent with those of the conventional serotyping (Table 2). No positive reaction was observed in any of the strains of the species Escherichia non-Burt.

TABLE 264 serum agglutination results of Escherichia burkitense and 8-fold PCR detection results

Example 3 characterization of multiplex PCR for Simultaneous detection of serotype of Escherichia albertiae O1-O7

1. Experiment of specificity

The specificity of the multiplex PCR method of the present invention was examined by detecting the following 151 different species of strains (Table 3). The detection shows that no non-specific amplification band exists, and the multiple PCR detection system for simultaneously detecting the serotype of the Escherichia coli O1-O7 has better species specificity.

TABLE 3 151 different species of bacteria used in the specificity test

2. Sensitivity test

The genomic DNA of the reference strain of each type was diluted in a gradient and used to test the sensitivity of the method. After extraction of genomic DNA as described above, 100. mu.L of DNA Rehydration Solution was added and dissolved overnight at 4 ℃. And (3) shaking and uniformly mixing the DNA template the next day, detecting the DNA concentration by using the Nanodrop and recording. Based on the DNA concentration, the DNA was serially diluted in multiple 6 concentrations of 1 ng/. mu.L, 100 pg/. mu.L, 50 pg/. mu.L, 20 pg/. mu.L, 10 pg/. mu.L and 1 pg/. mu.L, and 1. mu.L of each dilution was used as a template for the addition to the multiplex PCR reaction system, and the rest was as described above. The lower detection limit of the detection system is 10-50 pg.

3. Accuracy test

The accuracy of the multiplex PCR method disclosed by the invention is verified by using 64 strains of the Erbert Eichschka reference strain LMG20976, the feces isolated strain of a Shanghai diarrheal patient in 2013, and the 62 strains isolated from multiple sources in 2014-2016 in Sichuan province. The serotypes of these strains have all been identified by seroagglutination experiments. The results are shown in Table 2.

The results show that the serotype of the ebert ehrlichia reference strain LMG20976 is determined by a conventional serum agglutination experiment, one excrement isolate of a Shanghai diarrhea patient in 2013, and 62 strains separated from a plurality of sources in 2014-2016, province Sichuan, are identified by the multiplex PCR method, the coincidence rate of the two results is 100%, and the primer combination designed by the invention has excellent accuracy in multiplex PCR detection of 7 serotypes of the ebert ehrlichia.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

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Claims

1. A specific primer pair combination for simultaneously detecting serotypes of Escherichia coli O1-O7 is characterized by comprising the following primer pairs:

the nucleotide sequence of the specific primer pair for detecting the serotype of the Escherichia coli O7 is shown as SEQ ID NO. 13-14.

2. Use of a specific primer pair combination according to claim 1 for the preparation of a kit for the simultaneous detection of serotypes O1-O7 of escherichia coli.

3. The application of an internal reference primer in the preparation of a kit for detecting the serotype O1-O7 of Escherichia coli by combining and using the specific primer pair disclosed by claim 1, wherein the nucleotide sequence of the internal reference primer is shown as SEQ ID NO. 15-16.

4. A detection kit, characterized by comprising the specific primer pair combination of claim 1 and a pair of internal reference primers, wherein the nucleotide sequences of the internal reference primers are shown as SEQ ID NO. 15-16.

5. The kit of claim 4, wherein the operating program comprises the steps of:

(1) extracting nucleic acid of a sample to be detected;

(2) carrying out single-tube primary multiplex PCR reaction on sample DNA by using the specific primer pair combination of claim 1 and the internal reference primers shown in SEQ ID NO. 15-16; detecting the PCR product through electrophoresis, and judging whether the sample contains the Escherichia coli and which one of serotypes O1-O7 according to the fragment size of the PCR product;

6. The kit according to claim 5, wherein in the multiplex PCR, the concentrations of the primers in the PCR reaction solution are respectively as follows:

7. the kit of claim 5, wherein the multiplex PCR reaction conditions are: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 64 ℃, 60s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.

8. Use of a kit according to any one of claims 4 to 7 for the detection of the serotype of escherichia coli O1-O7 for non-diagnostic purposes.