LAMP (loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii and special primers of LAMP kit

A technology of Acinetobacter baumannii and a kit, which is applied in the biological field, can solve problems such as the absence of Acinetobacter baumannii, and achieve the effects of high sensitivity and good specificity suitable for large-scale popularization and application

Inactive Publication Date: 2015-03-25
INST OF PLA FOR DISEASE CONTROL & PREVENTION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no LAMP kits and special primers for the detection of Acinetobacter baumannii at home and abroad

Method used

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  • LAMP (loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii and special primers of LAMP kit
  • LAMP (loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii and special primers of LAMP kit
  • LAMP (loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii and special primers of LAMP kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Primer Design for LAMP Detection of Acinetobacter baumannii

[0042] The β-lactamase OXA-51 gene (bla OXA-51 ) sequence (GenBank No.: DQ385606.1), homology analysis was carried out by BLAST software, and the Acinetobacter baumannii β-lactamase OXA-51 gene (bla OXA-51 ) specific conserved sequence (SEQ ID NO: 1 in the sequence listing), and then according to the specific conserved sequence, use the software Primer design V4 to design primers for carrying out LAMP detection to Acinetobacter baumannii, and design five sets of primers , after experimental comparison, the primer combination OXA-4 was finally selected, and the primer sequences are shown in Table 1.

[0043] Table 1 is used for the primer combination OXA-4 of carrying out LAMP detection to Acinetobacter baumannii

[0044]

Embodiment 2

[0045] Embodiment 2, the establishment of the LAMP detection method of Acinetobacter baumannii

[0046] 1. Determine the optimal primer combination for the LAMP detection method of Acinetobacter baumannii

[0047] Use the five sets of primer combinations designed in Example 1 to perform LAMP detection on Acinetobacter baumannii (from the Department of Respiratory and Critical Care Medicine, Affiliated Hospital of the Academy of Military Medical Sciences) to obtain the best primer combination. The specific method is as follows:

[0048] 1) LAMP amplification was carried out under the guidance of the above five sets of primers respectively. The 25 μl LAMP reaction system included: Acinetobacter baumannii genomic DNA (Promega Genomic DNA Purification Kit A1125 to extract the nucleic acid in the sample to be tested) 2μl, 20mM Tris HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween20, 0.8M betaine (betaine), 8mM MgSO 4 , 1.4mM dNTP each, 8U Bst DNA polymerase, the amount...

Embodiment 3

[0057] Example 3, Specificity and Sensitivity Detection of the LAMP Detection Method of Acinetobacter baumannii

[0058] 1. Specific detection of the LAMP detection method for Acinetobacter baumannii

[0059]Acinetobacter baumannii (from the Department of Respiratory and Critical Care Medicine, Affiliated Hospital of the Academy of Military Medical Sciences), Acinetobacter hemolyticus (purchased from China General Microbiological Culture Collection Center (CGMCC)), hospital Acinetobacter (acinetobacter gene species TU13, purchased from CGMCC), Acinetobacter johnsoni (purchased from CGMCC), Acinetobacter junii (purchased from CGMCC), Pittii (acinetobacter gene species 3, purchased from China Industrial Microbial Culture Collection Management Center (China Center of Industrial Culture Collection, CICC), Acinetobacter calcoacetate (purchased from CGMCC), Acinetobacter lophia (stored by the Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy...

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Abstract

The invention discloses an LAMP (Loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii, special primers of the LAMP kit and an application of the LAMP kit to detection of the acinetobacter baumannii. The nucleotide sequence of the five special primers for performing LAMP detection for the acinetobacter baumannii in a sequence table is SEQ ID NO: 2(F3), SEQ ID NO: 3(B3), SEQ ID NO: 4(FIP), SEQ ID NO: 5(BIP) and SEQ ID NO: 6(LF). The acinetobacter baumannii can be rapidly, conveniently, efficiently, high-specifically and high-sensitively detected at the equal temperature, complicated instruments are omitted, and a novel technical platform for detecting the acinetobacter baumannii is provided. The LAMP kit can be used for screening and detecting the acinetobacter baumannii in livestock production units, basic medical and health units and disease prevention and control centers, has wide market prospects and high economic and social benefits and is suitable for wide-range popularization and application.

Description

technical field [0001] The invention belongs to the molecular biology detection method of bacteria in the field of biotechnology, in particular to a LAMP kit of Acinetobacter baumannii and its special primer and its application in detecting Acinetobacter baumannii. Background technique [0002] Current status of clinical infection and treatment of Acinetobacter baumannii [0003] Acinetobacter baumannii (Ab) is a non-fermenting, aerobic, Gram-negative bacilli belonging to the genus Acinetobacter. Acinetobacter baumannii mainly exists in the hospital environment and is one of the main pathogens of hospital-acquired pneumonia and ventilator-associated pneumonia. Patients in the intensive care unit, especially, have a higher probability of being infected with Acinetobacter baumannii. This is closely related to patients with low immunity, severe underlying diseases, invasive treatments such as ventilators, and extensive use of broad-spectrum antibacterial drugs. In addition, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2527/101
Inventor 袁静柏长青李璞媛刘威李环牛文凯邹大阳苑鑫刘慧莹
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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