Microwell plate for quantitatively detecting biotin with microbiological method, kit containing microwell plate and preparation method of microwell plate

A biotin and microplate technology, applied in the field of microplates, can solve the problems of strain loss, wrong results, high detection background, etc., and achieve the effect of improving accuracy and reducing strain loss.

Active Publication Date: 2017-04-26
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 plantarum. 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 biotin with microbiological method, kit containing microwell plate and preparation method of microwell plate
  • Microwell plate for quantitatively detecting biotin with microbiological method, kit containing microwell plate and preparation method of microwell plate
  • Microwell plate for quantitatively detecting biotin with microbiological method, kit containing microwell plate and preparation method of microwell plate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Microplate Preparation

[0054] (1) Activated Lactobacillus plantarum ATCC 8014

[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 plantarum ATCC 8014 strain into 10ml of medium, culture at 37±1°C until curdling, and store in the refrigerator at 4°C.

[0057] Add 0.3ml of the cultured bacteria in the above step to 10ml of culture medium, and cultivate at 37±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 plantarum ATCC 8014 strain into the Lactobacillus broth medium, culture at 36°C±1°C for 24h, centrifuge at 2100 rpm for 2min, stop the cultivation, and discard the supernatant;

[006...

Embodiment 2

[0074] 1. Microplate Preparation

[0075] Lactobacillus plantarum ATCC 8014 was used as the coated Lactobacillus plantarum 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, cultivate it at 38°C ± 1°C for 18h, and inoculate it at 2100°C. Centrifuge at rpm for 3 min to stop the culture and discard the supernatant. Add 197mM sucrose and 9mM CaCl 2 12ml of protective agent mixture, mix well, and then centrifuge for 2min, discard the supernatant, add 12ml of protective agent, mix well. Centrifuge as before and discard the supernatant. Add 10ml of protective agent and mix well. Aspirate 2ml of the bacterial suspension into 12ml of protective agent, mix well to make the test bacterial liquid.

[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 26mTo...

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 National Institute of Standards and Technology milk powder reference substance 1849C (biotin concentration is 199 μg / 100g)], do parallel experiments, the results are shown in Table 1. 1 dilution of milk powder reference substance 1849C was tested per batch (final concentration within the range of the standard curve).

[0090] Table 1

[0091]

[0092]

[0093] The coefficient of variation (1.04%) for determining the concentration of milk powder reference substance is very small, 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 milk powder reference substance 1849C [National Institute of Standards and Technology milk powder reference substance, concentration target value i...

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Abstract

The invention provides a microwell plate for quantitatively detecting biotin with a microbiological method, a kit and a preparation method of the microwell plate. By means of addition of a trehalose/saccharose and calcium chloride mixed liquid protective agent during preparing and improvement of a microbial solution drying method, microbial strain loss in the kit is reduced, and accuracy of a biotin detection result is improved. Viable counting proves that the number of active microbes preserved in lactobacillus plantarum dry foam in the microwell plate prepared with the method is 93%-95% of the total number of microbes in the added microbial solution while at least about 20% of microbes are lost with a common freeze-drying method.

Description

technical field [0001] The invention relates to a microporous plate for the quantitative detection of biotin by a microbiological method, a kit thereof and a preparation method thereof, belonging to the field of microbiological detection. Background technique [0002] At present, the detection methods of biotin at home and abroad are relatively complicated, generally including microbial method, high performance liquid phase method and enzyme-linked immunosorbent method. 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 bioactive biotin, which is the biggest feature and advantage of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12R1/25
CPCC12Q1/02
Inventor 刘龙飞高天垒卜庆婧
Owner BEIJING ZHONGJIAN BAOTAI BIOTECH
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