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A microporous plate for quantitative detection of folic acid by microbiological method, its kit and its preparation method

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

Active Publication Date: 2020-01-17
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 rhamnosus. 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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  • A microporous plate for quantitative detection of folic acid by microbiological method, its kit and its preparation method
  • A microporous plate for quantitative detection of folic acid by microbiological method, its kit and its preparation method
  • A microporous plate for quantitative detection of folic acid by microbiological method, its kit and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Microplate Preparation

[0054] (1) Activated Lactobacillus rhamnosus ATCC 7469

[0055] Add 10g of skimmed milk powder into 90ml of distilled water, mix well, divide into test tubes in units of 10ml, and sterilize at 115°C for 15 minutes. This solution is a liquid medium.

[0056] Add 0.2g of powdered Lactobacillus rhamnosus ATCC 7469 strain into 10ml of culture medium, culture at 38°C until curdling, and store in the refrigerator.

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

[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 rhamnosus ATCC 7469 strain into Lactobacillus broth medium, culture at 36°C±1°C for 24h, centrifuge at 2000rpm for 2min, stop the cultivation, and discard the supernatant;

[0061] Add 195...

Embodiment 2

[0074] 1. Microplate Preparation

[0075] Lactobacillus rhamnosus ATCC 7469 was used as the coated Lactobacillus rhamnosus strain, and the steps were as follows: Inoculate the activated strain (the activation treatment method is the same as that in Example 1) into the Lactobacillus broth medium, and cultivate at 38°C ± 1°C After 18 hours, centrifuge at 2000 rpm for 3 minutes to stop the culture, and discard the supernatant. Add 205mM sucrose and 10mM CaCl 2 Protective agent mixture 11ml, mix well, then centrifuge for 2min, discard the supernatant, add 11ml protective agent, mix well. Centrifuge as before and discard the supernatant. Add 9ml of protective agent and mix well. Aspirate 2ml of the bacterial suspension into 10ml of protective agent, mix well to make the test bacterial liquid.

[0076] Add 8 μl of the bacterial solution to each well of the microwell plate in equal parts, and heat the test bacterial solution slightly under the low vacuum environment of 26mTorr, 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 National Institute of Standards and Technology milk powder reference substance 1849B (folate concentration is 229.3 μg / 100g), do parallel experiments, the results are shown in Table 1. 1 dilution of the reference material was tested per batch (final concentration within the range of the standard curve).

[0090] Table 1

[0091]

[0092] The coefficient of variation (0.96%) for determining the concentration of milk powder reference substance 1849B is very small (0.96%), indicating high precision. The variation in the raw results of the standards among the 3 batches was less than 10%.

[0093] Use 3 batches of kits (kits prepared in Example 2) to detect National Institute of Standards and Technology milk powder reference substance 1849B (folate concentration is 229.3 μg / 100g), do parallel experiments, the resul...

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Abstract

The invention provides a microporous plate for quantitative detection of folic acid by a microbial method, wherein the microporous plate is prepared from the following steps: carrying out inoculation treatment of lactobacillus rhamnosus after being activated, and then adding a trehalose / sucrose and calcium chloride mixed liquid to prepare a test bacterial liquid; and then adding the test bacterial liquid to each hole of the microporous plate, heating the test bacterial liquid in a low vacuum environment, and finally carrying out drying treatment. The invention also provides a kit containing the microporous plate and a preparation method thereof. By addition of the trehalose / sucrose and calcium chloride mixed solution protective agent during preparation, and with an improved bacterial liquid drying method, the loss of bacteria in the kit is reduced, and the accuracy of a detection result of folic acid is improved. Through counting viable bacteria, the number of the viable bacteria preserved in lactobacillus rhamnosus dry foams in the microporous plate prepared by the method is more than 93% of the total number of bacteria in the added bacterial liquid (at least about 20% of bacteria is lost in a normal freeze drying method).

Description

technical field [0001] The invention relates to a microporous plate for the quantitative detection of folic acid 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 folic acid 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 folic acid, which is the biggest feature and advantag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02C12R1/225
CPCC12Q1/02
Inventor 刘龙飞高天垒卜庆婧
Owner BEIJING ZHONGJIAN BAOTAI BIOTECH
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