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Primer composition for detecting NDM-1 gene with high specificity and application thereof

A NDM-1, auxiliary detection technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effect of simple steps, self-safety and short reaction time

Inactive Publication Date: 2013-01-16
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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  • Primer composition for detecting NDM-1 gene with high specificity and application thereof
  • Primer composition for detecting NDM-1 gene with high specificity and application thereof
  • Primer composition for detecting NDM-1 gene with high specificity 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 primer composition for detecting a new metallo-beta-lactamase-1 (NDM-1) gene with high specificity and application thereof, and provides a special primer for detecting a coding gene of NDM-1 protein supplementarily, which consists of deoxyribonucleic acids (DNA) represented by sequences 1, 2, 3, 4, 5 and 6; and the NDM-1 protein is protein formed by an amino acid sequence represented by a sequence 7. The special primer has the characteristics of high sensitivity, speed and specificity, simplicity, convenience, safety and the like; and by using the special primer widely, the detectability of pathogenic microorganisms in apparatus collocation areas can be improved greatly, so the special primer has a great significance for the early-stage and accurate diagnosis of the pathogenic microorganisms. The special primer can guide rational administration of medicaments clinically to avoid delaying the state of an illness and increasing treatment cost, is favorable for controlling the propagation of bacteria and preventing outbreak and prevailing, and has a great significance for the improvement of treatment effects and the control of infection in hospitals.

Description

technical field [0001] The invention relates to a primer composition for detecting NDM-1 gene with high specificity 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 discovered for the first time the Methicillin-resistant Staphylococcus aureus (MRSA), which can secrete a phenol-soluble modulin (PSM) to destroy human tropism. Neutrophils, forming a serious infection-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 In...

Claims

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Application Information

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