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Preparation and application of rickettsia discrimination detection gene chip

A technology for the detection of gene chips and rickettsia, which is applied in the field of gene chip detection, can solve the problems of complex operation, detection of only one or several pathogens, and long time-consuming isolation and cultivation, etc., and achieves high specificity and good specificity Effect

Inactive Publication Date: 2015-05-27
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the isolation and culture takes a long time and the operation is complicated; PCR and immunological methods can only detect one or a few pathogens, which cannot fully meet the needs of detecting various types of Rickettsia

Method used

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  • Preparation and application of rickettsia discrimination detection gene chip
  • Preparation and application of rickettsia discrimination detection gene chip
  • Preparation and application of rickettsia discrimination detection gene chip

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Development of Gene Chip for Rickettsia Screening and Detection

[0040] 1. Primer probe design and screening

[0041] First, the Rickettsia target gene sequence was downloaded from the NCBI gene database. After the sequence was downloaded, the AlignX program in the Vector NTI Advance 10 (invitrogen) software package was used to perform a global alignment of the gene sequences of each pathogen according to the default parameter settings. Design specific oligonucleotide probes, general and specific primers at the conserved positions of the gene sequence according to the comparison results. After screening, a total of 12 upstream and downstream primers were finally determined, and the reverse primer was bio-labeled at the 5' end as a primer for the chip; 10 specific detection probes were determined, and NH at the 3' end 2 grooming.

[0042] 2. Preparation of oligonucleotide chip and probe array

[0043] After completing the probe screening, the final probe a...

Embodiment 2

[0051] Example 2: Determination of Positive Judgment Criteria for Rickettsia Screening Detection Gene Chip

[0052] The cutoff value is a criterion for judging whether the signal value of the gene chip is positive. Each typing probe selects non-rickettsia (i.e. negative bacterial strains) and blank control for gene chip hybridization, and through repeated experiments and data statistics, the background statistical average value +2SD of negative bacterial strains and blank control is used as each The cutoff value of the probe. The discrimination ability of each mutation detection probe was more than 2.5 times as the criterion for judging whether a mutation occurred at the site.

Embodiment 3

[0053] Example 3: Specificity Evaluation of Gene Chip for Rickettsia Screening and Detection

[0054] Specificity is the most important assessment index of a diagnostic method. The gene chip of the present invention uses optimized systems and conditions to detect Mycoplasma pneumoniae, Chlamydia pneumoniae, Staphylococcus aureus, Pseudomonas cepacia, Streptococcus pneumoniae, Pseudomonas aeruginosa Bacteria such as Haemophilus influenzae, Klebsiella pneumoniae and Stenotrophomonas maltophilia, by the attached Figure 4 It can be seen that the above bacterial test results were all negative for Rickettsia, with good specificity. The present invention detects 7 kinds of Rickettsia, by image 3 It can be seen that all 7 kinds of rickettsiae can be clearly distinguished, indicating that the specificity of the present invention is good.

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Abstract

The invention relates to preparation and application of a rickettsia discrimination detection gene chip. The preparation method comprises the following steps: preparing universal and specific primers, preparing seven rickettsia nucleic acid typing probes, preparing an oligonucleotide chip, establishing a multiplex PCR (polymerase chain reaction) system, and establishing a hybridization system. The gene chip can simultaneously discriminate seven important rickettsiae, including rickettsia prowazeki, rickettsia mooseri, spotted fever group rickettsia, Coxiella burnetii, Orientia tsutsugamushi, Ehrlichia chaffeensis and Anaplasma phagocytophilum. The gene chip has the advantages of high speed, high accuracy, high flux and high specificity, and can provide a novel detection means for clinical diagnosis and epidemiological investigation of rickettsia infection.

Description

technical field [0001] The invention relates to the preparation and application of a gene chip for rickettsia screening and detection, and belongs to the technical field of gene chip detection. Background technique [0002] Rickettsiae (rickettsiae) is a kind of obligate intracellular parasitic Gram-negative bacteria except for a very small number, and the rickettsiae caused by it is an important class of zoonotic natural foci diseases. It is sporadic and seasonal worldwide. Many arthropods such as lice, fleas, ticks, mites, etc. can be used as the transmission medium of Rickettsia infection. With the development of international tourism, rickettsial disease is considered to be one of the important infectious diseases that threaten the health of tourists. In recent years, the continuous emergence of new species of spotted fever and the prevalence of new Rickettsia such as Ehrlichia remind us of the possibility of large-scale epidemics and sudden outbreaks of rickettsial di...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C40B40/06C40B50/06C12R1/01
CPCY02A50/30
Inventor 王升启李灵云刘琪琦陈苏红张敏丽张英杰
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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