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Protein suspension chip method capable of quantitatively determining yersinia pestis

A technology of Yersinia pestis and suspension chips, which can be applied to measuring devices, biological testing, material inspection products, etc., can solve problems such as poor specificity, inconvenient nucleic acid detection methods, and cumbersome methods

Inactive Publication Date: 2009-07-29
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The indirect hemagglutination method (IHA) for detecting Yersinia pestis specific antibody (F1 antibody) has low sensitivity and poor specificity (Williams, J.E., Arntzen, L., Robinson, D.M. & et al.Comparison of passive haemagglutination and enzyme-linkedimmunosorbent assay for serodiagnosis of plague. Bull WHO 1982, 60, 777-781.)
For the nucleic acid detection method, although PCR technology is used to detect and analyze Yersinia pestis (Hinnebusch, J.&Schwan, T.G. New method for plague survival using polymerase chain reaction to detect Yersiniapestis infections. J Clin Microbiol 1993, 31 (6), 1511- 1514.; Higgins, J.A., Ezzell, J., Hinnebusch, B.J., Shipley, M., Henchal, E.A. & Ibrahim, M.S. 5'nuclease PCR assay to detect Yersinia pestis. JClin Microbiol 1998, 36(8), 2284-2288. ; Neubauer, H., Meyer, H., Prior, J., Aleksic, S., Hensel, A. & Splettstos ser, W. A combination of different polymerase chain reaction (PCR) as says for the presumptive identification of Yersinia pestis. J Vet MedB Infect Dis Vet Public Health 2000, 47(8), 573-580.), especially real-time fluorescent quantitative PCR technology (Herbert, T., Emil, C.R., Sascha, A.D.&etal.Rapid detection of Yersinia pestis with multiplex real- timePCR assays using fluorescent hybridisation probes. FEMS ImmunolMed Microbiol 2003, 38, 117-126), high specificity, not easy to be contaminated, but the methods of existing PCR technology are cumbersome, especially such methods include DNA / RNA extraction and complex operating steps, and cannot be used for the detection of non-nucleic acid substances such as proteins, so nucleic acid detection methods are limited and inconvenient

Method used

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  • Protein suspension chip method capable of quantitatively determining yersinia pestis
  • Protein suspension chip method capable of quantitatively determining yersinia pestis
  • Protein suspension chip method capable of quantitatively determining yersinia pestis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the preparation of the protein suspension chip that detects Yersinia pestis

[0055] 1. Capture antibody-coated encoded microspheres

[0056] First, the coded microspheres (Bead, microsphere) used in the present invention can be purchased from companies such as LUMINEX, BIO-RAD, etc. The coded microspheres used are used to label an antibody that can capture the corresponding target molecule, and the microspheres are coated with the corresponding antibody .

[0057]A. Activation of encoded microspheres

[0058] Take 100μL (1.25×10 6 pcs) encoded microspheres into a 1.5mL centrifuge tube, centrifuge at 14000g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere washing buffer to suspend, shake and sonicate, centrifuge at 14000g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere activation buffer, followed by 10 μL of freshly prepared EDC (50 mg / mL), followed by 10 μL of freshly prepared Sulfo-NHS (50 mg / mL), ...

Embodiment 2

[0072] Embodiment 2, the improvement of suspension chip preparation method condition

[0073] 1. Selection of microspheres coated with different antibodies and coating amount

[0074] Three kinds of monoclonal antibodies (2F6, 5G12, 2H12) and three kinds of polyclonal antibodies (rabbit anti-EV76, goat anti-EV76, rabbit anti-F1) against Yersinia pestis F1 were selected respectively, with 4 μg, 8 μg, 10 μg, 16 μg, 24 μg, 40 μg , The amount of 48 μg is coated with 100 μL of microspheres coded as No. 28.

[0075] After testing, 4 μg, 8 μg, 16 μg of monoclonal antibody 2F6, 48 μg of rabbit anti-EV76 and 40 μg of goat anti-EV76 coated microspheres were not effective, and could not meet the needs of detection after coating; 16 μg of monoclonal antibody 5G12, 24 μg of monoclonal antibody 2H12 and 16 μg rabbit anti-EV76, 10 μg goat anti-EV76 and 10 μg and 40 μg rabbit anti-F1 have good coating effect, and the MFI value is far greater than 2000. After counting under a microscope, stor...

Embodiment 3

[0088] Embodiment 3, preparation and detection of sample

[0089] 1. Preparation of samples to be tested

[0090] Prepare serial concentrations of F1 antigen standards to draw a standard curve for sample detection dose-response: dilute the F1 antigen with a 4-fold gradient with the sample diluent to form a serial concentration of the same sample for ELISA and suspension chip detection.

[0091] 2. Preparation of artificially polluted "white powder" samples

[0092] Mix a certain amount of F1 antigen into milk powder, starch, flour, instant fruit and other powder samples according to different concentrations, add 0.5g powder into 5mL sample dilution buffer, mix well, let stand for 2 hours, let the test The sample is fully adsorbed with the white powder.

[0093] For the above-mentioned contaminated "white powder" samples, choose cotton, thin filter paper, thick filter paper, 0.45μm filter membrane, 0.22μm filter membrane to filter, and 2000rpm, 5 minutes and 1000rpm, 4 minute...

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Abstract

The invention aims at providing a protein suspension chip which can quantitatively detect the bacteria and a detection method thereof, particulary relates to a method for detecting the protein suspension chip which is suitable for quantitative detection and analysis of Yersinia pestis (Y.pestis). The method has high sensitivity, strong specificity, good detection capacity and wide dynamic range, and establishes a new detection mode platform.

Description

technical field [0001] The invention relates to a method capable of quantitatively detecting bacteria, in particular to a protein suspension chip detection method suitable for quantitatively detecting and analyzing Yersinia pestis. Background technique [0002] Yersinia (Yersinia) Enterobacteriaceae, there are 11 species, three of which are pathogenic to humans, they are Yersinia pestis (Y. pestis), Yersinia pseudotuberculosis (Y. pseudotuberculosis) ) and Y. enterocolitica, hereinafter referred to as Yersinia pestis, pseudotuberculosis and enterocolitica, respectively. [0003] Among the three pathogenic Yersinia species, Yersinia pestis, Pseudotuberculosis bacteria and Enterocolitica bacteria, Yersinia pestis is the model species of Yersinia genus, it is Gram-negative coccal bacillus, does not move, does not form spores , Gimusa, Wright's or Wesson's staining is bipolar, and can grow in the range of 4-40°C, the optimum growth temperature is 28-30°C, the optimum growth pH ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/546
Inventor 王静孙肖红杨宇张晓龙胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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