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Dedicated primer for auxiliarily detecting NDM-1 (New Metallo-Beta-Lactamase-1) gene and application thereof

An NDM-1, auxiliary detection technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effects of simple steps, high sensitivity, and easy identification

Inactive Publication Date: 2013-04-03
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the detection of NDM-1 gene by LAMP method at home and abroad

Method used

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  • Dedicated primer for auxiliarily detecting NDM-1 (New Metallo-Beta-Lactamase-1) gene and application thereof
  • Dedicated primer for auxiliarily detecting NDM-1 (New Metallo-Beta-Lactamase-1) gene and application thereof
  • Dedicated primer for auxiliarily detecting NDM-1 (New Metallo-Beta-Lactamase-1) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, design and preparation of special primers

[0044] Each primer (FOP, BOP, FIP, BIP, FLP and BLP) shown in Table 1 was designed and synthesized.

[0045] The six primers shown in Table 1 form special primers and are used in Example 2.

[0046] Nucleotide sequence of table 1 primer

[0047]

[0048] The amino acid sequence of the NDM-1 protein is shown in sequence 7 of the sequence listing, its coding gene is shown in sequence 8 of the sequence listing, and the open reading frame is the 14th to 826th nucleotides from the 5' end of sequence 8 of the sequence listing.

Embodiment 2

[0049] Embodiment 2, application-specific primer detection NDM-1 gene

[0050] 1. Template preparation

[0051] The DNA (template) shown in the sequence 11 of the synthetic sequence list, in order to prevent the nucleotide sequence of the target sequence from being translated into an active functional protein, individual nucleotides in the target sequence were replaced, in order to facilitate the integration with For other template discrimination, the DNA shown in Sequence 11 is also called NDM-1 gene.

[0052] 2. Application of special primers to detect NDM-1 gene

[0053] 1. Interpret the results by observing the change of fluorescent color (fluorescence colorimetric method)

[0054] The template synthesized in Step 1 (concentration: 2.9 ng / μl) was used for LAMP (loop-mediated constant temperature amplification). The LAMP reaction system is shown in Table 2.

[0055] Table 2 LAMP reaction system (25μL)

[0056] components

The final concentration in the react...

Embodiment 3

[0086] Embodiment 3, the sensitive determination of special primer

[0087] 1. Prepare the template

[0088] The NDM-1 gene shown in the sequence 11 of the sequence listing was synthesized and used as a template after gradient dilution. The concentrations of the templates were as follows: 2.9×10 -6 ng / μl, 2.9×10 -7 ng / μl, 2.9×10 -8 ng / μl.

[0089] Second, the sensitivity of electrophoresis detection

[0090] The templates synthesized in Step 1 were used to carry out LAMP (loop-mediated constant temperature amplification), and the LAMP reaction system is shown in Table 3.

[0091] Use 2 μL of double distilled water as the template blank control, 2 μL of Klebsiella pneumoniae genomic DNA (82.8ng / μl) as negative control 1, and 2 μL of Escherichia coli genomic DNA (52.4ng / μl) as negative control 1 Negative control 2.

[0092]Mix the reagents in Table 3 except the template for use; pre-denature the template at 96°C for 3 minutes; then mix the template with other well-mixed r...

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Abstract

The invention discloses a dedicated primer for auxiliarily detecting a NDM-1 (New Metallo-Beta-Lactamase-1) gene and application thereof. The dedicated primer for auxiliarily detecting the NDM-1 (New Metallo-Beta-Lactamase-1) gene, provided by the invention, consists of DNA (deoxyribonucleic acids) shown by a sequence 1, a sequence 2, a sequence 3, a sequence 4, a sequence 5 and a sequence 6 in asequence table; and the NDM-1 protein is protein consisting of amino acid sequences shown in a sequence 7. The dedicated primer has the characteristics of high sensitivity, high speed, strong specificity, simpleness and convenience, safety, and the like while used for detection, can be widely applied to improve the pathogenic microorganism detection capability of a low apparatus location area, and has great significance for early and accurate diagnosis of pathogenic microorganisms. The dedicated primer can not only be used for guiding clinical reasonable medication and avoiding from delaying illness and increasing treatment expenses, but also is beneficial to controlling propagation of the pathogenic microorganisms and preventing outbreak, thereby having important significance for improving the treatment effect and controlling infection in hospitals.

Description

technical field [0001] The invention relates to a special primer for auxiliary detection of NDM-1 gene and application thereof. Background technique [0002] Bacteria started their own life course long before the birth of human beings. During World War II, bacteria seriously threatened human life. In 1929, Fleming accidentally discovered penicillin (Penicillin). With the advent of antibiotic drugs, bacterial drug resistance has been controlled in some countries. In 1961, British scholar Jevons first discovered methicillin-resistant Staphylococcus aureus (MRSA), which can secrete a phenol-soluble modulin (PSM) to destroy human Neutrophils, forming severe infections - the first "superbug" was born. In recent years, the dual factors of antibiotic abuse and antibiotic accumulation have led to the accumulation of drug resistance genes in bacteria, resulting in multi-drug resistance. [0003] According to the British journal "The Lancet Infectious Diseases" published on August ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N9/80C12N15/55
Inventor 朱宝利武娜张苑怡王志云
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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