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Detection reagent for Shigella flexneri plasmid-carried gene Ipt-O and application thereof

A technology of Shigella flexneri and lpt-o, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem that the antigenic determinant has not been discovered, and achieve simple judgment, avoid subjectivity, and accuracy The effect of identification

Active Publication Date: 2015-02-11
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the MASF IV-1 antigenic determinant has not yet been identified

Method used

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  • Detection reagent for Shigella flexneri plasmid-carried gene Ipt-O and application thereof
  • Detection reagent for Shigella flexneri plasmid-carried gene Ipt-O and application thereof
  • Detection reagent for Shigella flexneri plasmid-carried gene Ipt-O and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, the design of lpt-O gene-specific detection primer

[0061] 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 1 In b, it has a typical X serotype O antibody structure (Kenne et al., 1977) ( figure 2 Middle B), in addition, in the NMR spectrum of Xv bacterial strain, find the signal of a PEtN group (Table 1), this PEtN group adds in RhaII ( figure 2 Middle B), and this is the same as serotype X ( figure 2 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.

[0062] Table 1 1 H and 13 C NMR chemical shift s(δ, ppm)

[0063]

[0064] In the present invention, a gene SFxv_5135 and PEtN transferase protein Lpt3(N.meningitides), LptA(N.meningitides), L...

Embodiment 2

[0071] Embodiment 2, PCR amplification detection distinguishes Xv and X serotype bacterial strain

[0072] In this example, 10 strains of Shigella flexneri serotype Xv and 5 strains of X serotype were detected by using the designed primer pair lpt-O-2.

[0073] PCR amplification:

[0074] The primer pair was commissioned to Sangon Biotech (Shanghai) for synthesis. PCR amplification was carried out using a PCR amplification kit from TaKaRa Company (KakaRa, Japan). Each PCR reaction mixture contains: 1×PCR buffer, 0.2 μM of primers, 3 μl of template DNA, 2.5U DNA polymerase and 0.4 mM dNTP, total 50 μl. PCR amplification was carried out using PCR instrument SensoQuest LabCycler (Germany), and the specific conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 50 s, and extension at 72°C for 2 min, a total of 30 cycles; finally, extension at 72°C for 5 min.

[0075] The amplified products were subjected to electrophoresis, and...

Embodiment 3

[0076] Embodiment 3, PCR amplification detects MASF IV-1 phenotype

[0077] In the present embodiment, according to the amplification method described in Example 2, the 333 strains listed in Table 2 include 15 serotype strains for PCR amplification detection, and the results are only positive in MASF IV-1 strains (26Xv, 1 strain 4av and 25 strains Yv) and 4 strains X, 1 strain 4b, confirmed by sequencing, a single base deletion mutation occurred in the lpt-O gene in these 4 strains X and 1 strain 4b (Table 2) ( Figure 7 , Figure 8 ).

[0078] Carrying situation of table 2 lpt-O gene in Shigella flexneri

[0079]

[0080]

[0081] 1 + and - / + indicate the presence of the lpt-O gene. The number of positive strains is shown in parentheses.

[0082] 2 4av serotype strains (2002091 and NCTC 8296) and 4a serotype strains were differentially agglutinated in MASF IV-1.

[0083] 3 The serotype difference between Yv serotype strains and Y serotype strains lies in MASF IV-1 ...

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Abstract

The invention relates to a detection reagent for a Shigella flexneri plasmid-carried gene Ipt-O and an application thereof, and in particular relates to an application of the detection reagent for a Shigella flexneri plasmid-carried gene Ipt-O in preparation of a Shigella flexneri serotype detection agent. The detection agent is used for detecting the Shigella flexneri serotype or detecting the differential diagnosis sample containing Shigella flexneri. The invention also relates to a reagent for Shigella flexneri MASF IV-1 phenotype detection, which is composed of a primer pair of the Shigella flexneri plasmid-carried gene Ipt-O as shown in the SEQ ID No.2 and SEQ ID No.3, and further relates to a method for detecting the Shigella flexneri MASF IV-1 phenotype by using the primer pair, and a method for distinguishing Shigella flexneri Xv serotype and X serotype, 4a serotype and 4av serotype as well as Y serotype and Yv serotype.

Description

technical field [0001] The invention relates to the field of biotechnology detection of Shigella flexneri serotypes, in particular to the detection of the gene lpt-O (LPS phosphoethenolamine transferase for O-antigen, O-anti-lipopolysaccharide phosphoethanolamine transferase) carried by the Shigella flexneri plasmid Reagents and applications thereof, in particular to detection primers for the gene lpt-O carried by Shigella flexneri plasmids and their use in identifying the MASF IV-1 phenotype of Shigella flexneri. Background technique [0002] Shigella flexneri, the major causative agent of bacillary dysentery in developing countries, is estimated to infect approximately more than one million people each year and cause many deaths, most of them children under the age of five (Kotloff et al. , 1999). [0003] There is ample evidence that human immunity to dysentery infection is serotype-specific (Phalipon et al., 1995). According to the difference of lipopolysaccharide (LPS...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/10
Inventor 孙强正王建平徐建国
Owner ICDC CHINA CDC
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