Simple kit for detecting change index of short-chain fatty acid in excrement

Abstract

The invention relates to the technical field of kits, and discloses a simple kit for detecting change indexes of short-chain fatty acid in excrement, which comprises a sterile collection rod, a freeze-drying reaction tube, a standard colorimetric card, a portable colorimeter and a mobile phone APP auxiliary tool. According to the simple kit for detecting the change index of the short-chain fatty acid in the excrement, about 0.5 g of middle-section excrement is taken through the collecting rod and inserted into the reaction tube, and a clarified extracting solution is obtained through dissolving of the pre-extracting solution and filtering of the built-in filter membrane. The short-chain fatty acid in the extracting solution and 2, 4-dinitrophenylhydrazine are subjected to a derivatization reaction under the acidic condition to generate an orange red hydrazone compound, and a detection result and an interpretation suggestion are obtained through comparison with naked eyes in a colorimetric card, photographing analysis by a mobile phone APP or result interpretation by a portable colorimeter. The method improves the accuracy and real-time dynamics of detection, can be used for evaluating the effect of probiotic and diet intervention, and is beneficial to realizing precise medical treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of kits, and more specifically, it relates to a simple kit for detecting the change index of fecal short-chain fatty acids. Background Art

[0002] Short-chain fatty acids (SCFAs), including acetic acid, propionic acid, and butyric acid, are metabolites produced by the fermentation of dietary fiber by the gut microbiota. They play roles in regulating energy metabolism, enhancing insulin sensitivity, inhibiting lipogenesis and promoting lipolysis, and are involved in the regulation of gut microbiota-host interaction and appetite and feeding behavior regulation. They play an important role in the occurrence and development of obesity and show potential therapeutic value.

[0003] However, traditional SCFAs detection techniques have some limitations, mainly reflected in low sensitivity, poor specificity, and operational complexity. For example, although chromatography is accurate, it is complex to operate and costly, and spectrometry is fast but not sensitive enough. In addition, there are problems with the complexity, volatility, and stability of sample pretreatment. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a simple kit for detecting the change index of fecal short-chain fatty acids.

[0005] The present invention provides a simple kit for detecting the change index of fecal short-chain fatty acids, including a sterile collection stick, a freeze-dried reaction tube, a standard colorimetric card, a portable colorimeter, and a mobile phone APP auxiliary tool. The method of using this kit includes the following steps:

[0006] S1, Sample collection and processing;

[0007] S101, Sampling;

[0008] Use the collection stick to take about 0.5 g of middle-section feces and insert it into the reaction tube;

[0009] S102, Dissolution;

[0010] Add 1 ml of pre-loaded extraction solution (methanol: water = 4:1), shake for 10 seconds and then stand still for 1 minute to allow the sample to react fully with the pre-loaded extraction solution;

[0011] S103, Filtration;

[0012] Pass the liquid with complete reaction through the built-in filter membrane (0.45 μm) to remove solid particles and obtain a clarified extraction solution;

[0013] S2, Color reaction;

[0014] S201, Activation reaction;

[0015] Pour all of the filtered extract into a freeze-drying reaction tube, immediately tighten the tube cap. The pH of the reaction solution in the tube is 2.0, which can inhibit the interference of long-chain fatty acids (LCFAs) and non-carboxylic substances (such as sugars).

[0016] S202, Incubate;

[0017] Rub the reaction tube between the palms for 10 seconds, then place it in an environment at 37°C (such as body temperature or a heat-insulating bag) for 15 minutes to avoid color instability caused by high temperature. At this time, short-chain fatty acids (SCFAs) continuously react with 2,4-dinitrophenylhydrazine to form orange-red hydrazone compounds;

[0018] S203, Terminate the reaction;

[0019] Add 1 drop of the termination solution (10% NaOH) and shake well until the color stabilizes;

[0020] S3, Result interpretation;

[0021] Method 1 (Visual comparison with a colorimetric card): Pour the reaction solution into a transparent cuvette and compare it with the standard colorimetric card under natural light to select the color grade that is closest;

[0022] Method 2 (Analysis by taking pictures with a mobile phone APP): Take a picture of the reaction tube on a white background, and the APP automatically recognizes the RGB values and converts them into concentrations;

[0023] Method 3 (Portable colorimeter): Insert the colorimeter, and after 5 seconds, the results of three grades of "low (<20 μM) / medium (20 - 40 μM) / high (>40 μM)" will be displayed;

[0024] The beneficial effects of the present invention are as follows: By detecting the content and type changes of SCFAs in feces to characterize the improvement of the intestinal flora of the human body (including other animals), dynamic monitoring can be achieved. Moreover, the detection range of the kit covers the common concentration range (5 - 100 μM) of human (including other animals') feces, and the lower detection limit is low, enabling the identification of mild SCFAs deficiency.

[0025] It has stronger specificity than traditional detection methods, can avoid the influence of common components in feces, and has no significant interference when the bile salt is <1 μM and the glucose is <5%. The within-batch precision is high, CV < 8% (n = 3), and the stability of the lyophilized agent is good, and it can be stored for 18 months under the conditions of being closed and light-proof at 2 - 8°C.

[0026] The operation complexity is simple and the cost is low: The concentration of SCFAs in feces is significantly higher than that in blood or urine, and it can be directly detected without complex derivatization. Moreover, the sample does not require fasting or special preparation, the collection is painless and simple, and no other equipment is required during the detection process. Description of the Drawings

[0027] Figure 1is the status of short-chain fatty acids reflected by the colorimetric results of the present invention;

[0028] Figure 2 is the reaction color gradient table of the DNPH kit of the present invention;

[0029] Figure 3 is the picture of the standard colorimetric card of the present invention. Detailed implementation manners

[0030] Now, the subject matter described herein will be discussed with reference to exemplary embodiments. It should be understood that discussing these embodiments is only to enable those skilled in the art to better understand and thus implement the subject matter described herein. Without departing from the scope of protection of the content of this specification, changes can be made to the functions and arrangements of the elements discussed. Each example can omit, substitute, or add various processes or components as needed. Additionally, the features described in some examples can also be combined in other examples.

[0031] In at least one embodiment of the present invention, a simple kit for detecting the change index of fecal short-chain fatty acids is disclosed, including a sterile collection stick, a freeze-dried reaction tube, a standard colorimetric card, a portable colorimeter, and a mobile phone APP auxiliary tool. The method of using this kit includes the following steps:

[0032] S1, Sample collection and processing;

[0033] S101, Sampling;

[0034] Use the collection stick to take about 0.5 g of middle-section feces and insert it into the reaction tube;

[0035] S102, Dissolution;

[0036] Add 1 ml of pre-loaded extraction solution (methanol: water = 4:1), shake for 10 seconds and then stand still for 1 minute to allow the sample to fully react with the pre-loaded extraction solution;

[0037] S103, Filtration;

[0038] Pass the liquid with complete reaction through the built-in filter membrane (0.45 μm) to remove solid particles and obtain a clarified extraction solution;

[0039] S2, Color reaction;

[0040] S201, Activation reaction;

[0041] Pour all the filtered extraction solution into the freeze-dried reaction tube, immediately tighten the tube cap. The pH of the reaction solution in the tube is 2.0, which can inhibit the interference of long-chain fatty acids (LCFAs) and non-carboxylic substances (such as sugars);

[0042] S202, Incubation;

[0043] Rub the reaction tube with the palm for 10 seconds, and place it in an environment of 37 °C (such as body temperature or a thermal insulation bag) for 15 minutes. Avoid high temperature causing unstable color development. At this time, short-chain fatty acids (SCFAs) continuously react with 2,4-dinitrophenylhydrazine to generate orange-red hydrazone compounds;

[0044] Add S203 to terminate the reaction;

[0045] Add 1 drop of terminating solution (10% NaOH), and shake well until the color is stable;

[0046] Step S3, result interpretation;

[0047] Scheme 1 (visual comparison with a colorimetric card): Pour the reaction solution into a transparent colorimetric cell, and compare it with the standard colorimetric card under natural light to select the color grade that is closest.

[0048] Scheme 2 (mobile APP photo analysis): Place the reaction tube on a white background and take a photo. The APP automatically recognizes the RGB value and converts it into a concentration.

[0049] Scheme 3 (portable colorimeter): Insert the colorimeter, and after 5 seconds, the results of three grades of "low (<20 μM) / medium (20 - 40 μM) / high (>40 μM)" will be displayed.

[0050] Appendix Figure 1 shows the short-chain fatty acid status reflected by the colorimetric results. Appendix Figure 2 is the reaction color gradient table of the DNPH kit. Marked appendix Figure 3 is a picture of the standard colorimetric card.

[0051] The above describes the embodiments of the present invention. However, these embodiments are not limited to the above specific implementation manners. The above specific implementation manners are only illustrative and not restrictive. Under the inspiration of this embodiment, those of ordinary skill in the art can also make more equivalent embodiments in various forms, all of which fall within the protection scope of this embodiment.

Claims

1. A simple kit for detecting changes in fecal short-chain fatty acids, comprising a sterile collection stick, a freeze-dried reaction tube, a standard colorimetric card, a portable colorimeter, and a mobile phone APP auxiliary tool, characterized in that: The method of using the kit comprises the following steps: S1, sample collection and processing; S101, sampling; Use a collection stick to collect about 0.5g of mid-section feces and insert it into the reaction tube; S102, dissolution; Add 1 ml of pre-loaded extract solution (methanol: water = 4:1), shake well for 10 seconds and let stand for 1 minute to allow the sample to fully react with the pre-loaded extract solution; S103, filtration; The liquid after the reaction is completely passed through a built-in filter membrane (0.45 μm) to remove solid particles and obtain a clarified extract; S2, color reaction; S201, activation reaction; Pour all the filtered extract into the freeze-drying reaction tube and tighten the tube cap immediately. The pH of the reaction solution in the tube is 2.0, which can inhibit the interference of long-chain fatty acids (LCFAs) and non-carboxylic acid substances (such as sugars); S202, incubation; Rub the reaction tube with your palm for 10 seconds and place it in a 37°C environment (such as body temperature or a heat preservation bag) for 15 minutes to avoid color instability caused by high temperature. At this time, short-chain fatty acids (SCFAs) react with 2,4-dinitrophenylhydrazine to form orange-red hydrazone compounds. S203, termination reaction; Add 1 drop of stop solution (10% NaOH) and shake until the color is stable; S3, result interpretation; Option 1 (visual comparison with colorimetric card): Pour the reaction solution into a transparent colorimetric dish, compare it with the standard colorimetric card under natural light, and select the closest color level. Solution 2 (Photo analysis using mobile phone APP): Place the reaction tube on a white background and take a photo. The APP will automatically identify the RGB value and convert the concentration. Option 3 (portable colorimeter): Insert the colorimeter, and after 5 seconds, three levels of results will be displayed: "Low (<20μM) / Medium (20-40μM) / High (>40μM)".

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