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Primers for serotype detection of Shigella flexneri and multiplex amplification using the primers

A technology for Shigella flexneri and serotype detection, applied in the biological field, can solve the problems of expensive antiserum reagents, errors, and inability to effectively distinguish

Active Publication Date: 2015-09-30
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this multiplex amplification assay does not distinguish between Xv and X serotypes
In this prior art, when distinguishing between Xv and X serotypes, the traditional IV antiserum slide agglutination reaction still needs to be further adopted, and the slide agglutination method is time-consuming, the antiserum reagents used are expensive, and the results judged by visual inspection will lead to erroneous results
In addition, the method disclosed in CN102329861A cannot effectively distinguish the newly discovered Yv and Y serotypes, as well as 4a and 4av serotypes

Method used

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  • Primers for serotype detection of Shigella flexneri and multiplex amplification using the primers
  • Primers for serotype detection of Shigella flexneri and multiplex amplification using the primers
  • Primers for serotype detection of Shigella flexneri and multiplex amplification using the primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The determination of the determinant lpt-O gene of embodiment 1, MASF IV-1 antigen and the design of specific detection primer

[0049] In this embodiment, two-dimensional (two-dimensional) 1 H, 1 H and 1 H, 13 C NMR technology (Duus et al., 2000), analyzed the LPS structure of Xv serotype strain 2002017, and its 1 H NMR and 13 C NMR spectrum see figure 2 In b, it has a typical X serotype O antibody structure (Kenne et al., 1977) ( image 3 Middle B), in addition, in the NMR spectrum of Xv bacterial strain, find the signal of a PEtN group (Table 3), this PEtN group is added in Rha II ( image 3 Middle B), and this is the same as serotype X ( image 3 The only difference in A). Considering the serotype difference between the two, it can be judged that PEtN modification is the reason for the appearance of MASF IV-1+ phenotype.

[0050] table 3 1 H and 13 C NMR chemical shift s(δ, ppm)

[0051]

[0052]In this example, further through genome comparison, in...

Embodiment 2

[0068] Embodiment 2, Shigella flexneri serotype detection involving multiple amplification primers and multiple amplification

[0069] Materials and methods

[0070] Strains: In this example, 18 known and atypical serotypes (see Table 5) of Shigella flexneri were used for the establishment of multiplex PCR method conditions. A total of 390 Shigella flexneri (Table 6) were used for the evaluation of the effectiveness of the multiplex PCR method of the present invention. To detect the cross-reaction of the primers in this study, 38 strains of different genera (S. Sonnei (n=2), S. dysenteriae (n=12, including all 12 serotypes), S. boydii (n=18, including all 18 serotypes), EHEC (EDL933), E. coli (HB101), E. coli K12 (MG1655), Listeria monocytogenes (54003), etc. Serotypes of all strains of Flexneri Through multivalent antiserum (purchased from Denka Seiken, Japan) and monoclonal antibody (purchased from Reagensia AB, Sweden) verification.All Chinese bacterial strains used in th...

Embodiment 3

[0092] Embodiment 3, specific detection

[0093] In order to evaluate the specificity of the primers, this example refers to the method described in Example 2, and uses the 9 pairs of primers to perform multiple amplification (wherein the annealing temperature is 55°C), and detects 38 non-Fowleria strains, including other Shiga Groups and enteric pathogens. These bacteria amplified negatively (see Figure 13 , Table 8). The specificity of the method of the present invention was proved to be 100%.

[0094] Table 8 is used for the bacterial strain of specificity analysis and PCR amplification result

[0095] Strain / serotype

Number of strains

Specific gene PCR reaction results

S. Sonnie (S)

1

-

S. Sonnie(R)

1

-

S. dysenteriae (1-18)

Each 1

-

S. boydii(1-12)

Each 1

-

EHEC (EDL933)

1

-

UPEC (CFT073)

1

-

Escherichia coli K12 (HB101)

1

-

...

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Abstract

The invention relates to a Shigella flexneri serotype detection primer, and multiplex amplification using it. The primer comprises sequences represented by SEQ ID Nos.2 and 3, SEQ ID Nos.4 and 5, SEQ ID Nos.6 and 7, SEQ ID Nos.8 and 9, SEQ ID Nos.10 and 11, SEQ ID Nos.12 and 13, SEQ ID Nos.14 and 15, SEQ ID Nos.16 and 17, SEQ ID Nos.18 and 19. The primer is specific and has an annealing temperature consistency. The invention also relates to a method for realizing the multiplex amplification by using the primer, and further relates to an application of the Shigella flexneri serotype detection primer in the preparation of a detection agent, and a Shigella flexneri serotype detection kit including the primer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to primers for detection of Shigella flexneri and multiple amplification using the primers. Background technique [0002] Shigella is the main pathogen of bacterial diarrhea in developing countries. Each year, 164.7 million people worldwide are infected, resulting in 110,000 deaths, mostly children under the age of 5 (Kotloff, K.L., J.P. Winickoff, B. Ivanoff, J.D. Clemens, D.L. Swerdlow, P.J. Sansonetti, G.K. Adak, and M.M. Levine. 1999. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 77:651-66). Of the four groups of Shigella, Flexneri is the predominant group affecting the poor. [0003] According to the structure of O-antibody, Shigella flexneri is divided into different serotypes. So far, at least 16 serotypes have been reported, namely 1a, 1b, 1c, 1d, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
CPCC12Q1/04C12Q1/686C12Q2537/143C12Q2531/113
Inventor 孙强正王建平徐建国
Owner ICDC CHINA CDC
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