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Microwell plate for quantitatively detecting vitamin B12 by microbiological method as well as kit and preparation method of microwell plate

A vitamin and B12 technology, applied in the field of microwell plates, can solve problems such as strain loss, wrong results, high detection background, etc., and achieve the effect of improving accuracy and reducing strain loss

Active Publication Date: 2017-02-22
BEIJING ZHONGJIAN BAOTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are many defects in the kit coated with Lactobacillus delbrueckii. The existing method freezes the microbial suspension in the microwell plate at -80°C, and then freezes the microbial mass in the microwell plate under vacuum.
Some microorganisms mutate or recombine to adapt to unfavorable environments, so that after adding water or samples, some microorganisms can still grow in the absence of vitamins, resulting in high detection background and, in individual cases, erroneous results
In addition, existing methods using freeze-drying under vacuum can also result in at least about 20% loss of the strain

Method used

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  • Microwell plate for quantitatively detecting vitamin B12 by microbiological method as well as kit and preparation method of microwell plate
  • Microwell plate for quantitatively detecting vitamin B12 by microbiological method as well as kit and preparation method of microwell plate
  • Microwell plate for quantitatively detecting vitamin B12 by microbiological method as well as kit and preparation method of microwell plate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Microplate Preparation

[0054] (1) Activated Lactobacillus delbrueckii ATCC 7830

[0055] Add 11g of skimmed milk powder to 89ml of distilled water, mix well, dispense into test tubes in units of 10ml, and sterilize at 115°C for 15 minutes. This solution is a liquid medium.

[0056] Add 0.3g of powdered Lactobacillus delbrueckii ATCC 7830 strain into 10ml of medium, culture at 36±1°C until curdling, and store in the refrigerator.

[0057] Add 0.3ml of the bacteria cultured in the above step to 10ml of culture medium, and cultivate at 36±1°C until curdling.

[0058] Repeat the previous step several times until the curd time is stable, indicating that the bacteria have been activated.

[0059] (2) Preparation of test bacteria solution

[0060] Inoculate the activated Lactobacillus delbrueckii ATCC 7830 strain into the Lactobacillus broth medium, culture at 36°C±1°C for 24h, centrifuge at 2000rpm for 2min, stop the cultivation, and discard the supernatant;

[0061]...

Embodiment 2

[0074] 1. Microplate Preparation

[0075] Lactobacillus delbrueckii ATCC 7830 was used to coat the Lactobacillus delbrueckii strain, and the steps were as follows: Inoculate the activated bacterial strain (the activation treatment method is the same as in Example 1) into the Lactobacillus broth medium, and cultivate it at 38°C ± 1°C for 18h, Centrifuge at 2000 rpm for 3 min to stop the culture and discard the supernatant. Add 190mM sucrose and 9mM CaCl 2 Mix 10ml of protective agent mixture, centrifuge for 2min, discard the supernatant, add 10mL of protective agent, and mix evenly. Centrifuge as before and discard the supernatant. Add 10mL of protective agent and mix well. Aspirate 2ml of the bacterial suspension into 10mL of protective agent, mix well to make the test bacterial solution.

[0076] Add 1 μl of the bacterial solution to each well of the microwell plate, and heat the bacterial solution slightly under the low vacuum environment of 25mTorr, 50mTorr, 500mTorr, 5...

experiment example 1

[0088] This experimental example is to study the precision of the kit of the present invention.

[0089] Use 3 batches of kits (kits prepared in Example 1) to detect NIST reference substance 1849A [National Institute of Standards and Technology milk powder reference substance, the concentration target value is 4.82 μg / 100g], do parallel experiments, the results are shown in Table 1 . 1 dilution of NIST reference material 1849A was tested per batch (final concentration within the range of the standard curve).

[0090] Table 1

[0091]

[0092]

[0093] The coefficient of variation for determining the concentration of the NIST reference substance was very small (0.81%), indicating high precision. The variation in the raw results of the standards among the 3 batches was less than 10%.

[0094] Use 3 batches of kits (kits prepared in Example 2) to detect NIST reference substance 1849A [National Institute of Standards and Technology milk powder reference substance, concent...

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Abstract

The invention provides a microwell plate for quantitatively detecting vitamin B12 by a microbiological method as well as a kit and a preparation method of the microwell plate. According to the invention, a trehalose / saccharose and calcium chloride mixed liquid protecting agent is added in preparation, and a drying method for improving bacteria liquid is used, so that the strain loss in the kit is reduced, and the accuracy of a vitamin B12 detection result is improved. By counting viable bacteria, the number of active strains stored in Lactobacillus delbrueckii drying foam in the prepared microwell plate prepared by the method exceeds 90 percent of the total number of strains in added bacteria liquid (at least about 20 percent of strains are lost by an ordinary freeze drying method).

Description

technical field [0001] The invention relates to a microporous plate for the quantitative detection of vitamin B12 by a microbiological method, a kit and a preparation method thereof, and belongs to the field of microbiological detection. Background technique [0002] At present, the detection methods for vitamin B12 at home and abroad are relatively complicated, generally including microbial methods, high performance liquid chromatography and enzyme-linked immunosorbent assays. Among them, the lower limit of HPLC detection is much higher than that of microbial method, and the accuracy is better, but it requires expensive precision instruments and reagents, and the sample processing process is relatively complicated, so it is difficult to popularize and apply in production; in addition, enzyme-linked immunoassay detection The result is quite different from the actual value. The microbial method can sensitively detect the biologically active vitamin B12, which is the biggest ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06
CPCC12Q1/06
Inventor 刘龙飞高天垒卜庆婧
Owner BEIJING ZHONGJIAN BAOTAI BIOTECH
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