CN-122012758-A - Quantitative detection method for caproic acid bacteria

Abstract

The invention belongs to the field of microorganism and molecule detection, and particularly relates to a quantitative detection method of caproic acid bacteria. Aiming at the problems of long detection time, complicated operation steps, low flux, high requirement on strict anaerobic conditions, easiness in infection of infectious microbe and the like of the traditional detection method, the novel detection method for caproic acid bacteria is provided, a qPCR specific primer pair is designed based on the recA gene of caproic acid bacteria to be detected, and the qPCR specific primer pair is utilized to detect caproic acid bacteria. The primer pair and the method provided by the invention can effectively improve the identification efficiency of caproic acid bacteria, optimize the caproic acid bacteria screening method and provide a quicker detection method for caproic acid bacteria related research.

Inventors

• TAO YONG
• LIU TONG
• ZHANG LIXIA
• WANG YAN
• ZHANG YANYAN
• HE XIAOHONG
• YUAN SIQI

Assignees

• 中国科学院成都生物研究所
• 四川轻化工大学

Dates

Publication Date

20260512

Application Date

20260206

Claims (10)

1. 1. The primer pair is characterized by comprising an upstream primer P4f and a downstream primer P4r, wherein the sequence of the upstream primer P4f is shown as SEQ ID NO. 8, and the sequence of the downstream primer P4r is shown as SEQ ID NO. 9.
2. 2. A detection reagent, test strip or kit comprising the primer pair of claim 1.
3. 3. Use of the primer pair of claim 1 or the detection reagent, test paper or kit of claim 2 for detecting caproic acid bacteria.
4. 4. A method for detecting caproic acid bacteria is characterized in that a qPCR specific primer pair is designed based on recA genes of caproic acid bacteria to be detected, and the qPCR specific primer pair is utilized to detect caproic acid bacteria.
5. 5. The method of claim 4, wherein the primer pair comprises an upstream primer P4f with a sequence shown in SEQ ID NO. 8 and a downstream primer P4r with a sequence shown in SEQ ID NO. 9.
6. 6. The method according to claim 4 or 5, characterized in that the method comprises the steps of: (1) Designing a qPCR specific primer pair based on recA genes of caproic acid bacteria to be detected; (2) And extracting total DNA of the sample to be detected, carrying out qPCR amplification detection by using the primer pair, judging that the sample contains caproic acid bacteria to be detected if a strip appears, and judging that the sample does not contain caproic acid bacteria to be detected if the strip does not appear.
7. 7. The method according to claim 6, wherein the qPCR amplification reaction system of 10. Mu.L comprises 2X SsoAdvanced Universal SYBR Green Supermix. Mu.L, 0.2. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the standard plasmid template, 3.6μL。
8. 8. The method of claim 6, wherein the qPCR amplification reaction sequence comprises 95℃pre-denaturation for 3min, 95℃denaturation for 15s,60℃annealing for 30s, and 40 cycles.
9. 9. The method according to claim 6, wherein the method further comprises the steps of (3), (4): (3) Constructing a standard curve: (3-1) plasmid concentration measurement and copy number calculation, namely measuring the concentration of a standard plasmid and calculating the copy number; (3-2) drawing a standard curve, namely taking the logarithm of the copy number of the standard plasmid as an abscissa and the Ct value as an ordinate, generating the standard curve, and calculating a regression equation and a correlation coefficient R2; (4) And quantitatively detecting caproic acid bacteria, namely substituting the Ct value of the sample to be detected into the standard curve regression equation, and calculating the copy number of the gene recA in the sample to be detected, so as to obtain the cell content of the caproic acid bacteria to be detected.
10. 10. The method of claim 8, wherein the copy number is calculated by the formula copy number = Copy number units are copies/. Mu.L, and the total length units of the plasmid are bp.

Description

Quantitative detection method for caproic acid bacteria Technical Field The invention belongs to the field of microorganism and molecule detection, and particularly relates to a quantitative detection method of caproic acid bacteria. Background Caproic acid bacteria are key functional microorganisms in brewing of strong aromatic Chinese spirits, caproic acid generated by metabolism of caproic acid bacteria reacts with ethanol to generate ethyl caproate, and the caproic acid bacteria are main components forming aroma of main bodies of the strong aromatic Chinese spirits. The abundance of caproic acid bacteria in the pit mud is closely related to the quality of the pit mud and the flavor quality of the white spirit. Therefore, the real-time monitoring of the change of the abundance of caproic acid bacteria is of great importance to process control, and especially when the system is impacted or unstable in operation, timely grasping the dynamics of the flora is helpful for maintaining the operation stability and guaranteeing the treatment efficiency. However, as a plurality of microorganisms exist in the complex environment of white spirit brewing, caproic acid bacteria cannot be distinguished and quantified directly by a conventional plate counting method, which severely restricts effective monitoring of flora change in a pit or a wastewater system, thereby influencing the accuracy and application effect of actual regulation. Furthermore, since caproic acid bacteria are mostly obligate anaerobic bacteria, they are difficult to culture, slow to grow and poor in reproducibility in conventional agar plates, which further brings a barrier to quantitative detection of caproic acid bacteria. In view of the limitations of long detection time, complicated operation steps, low flux, high requirements on strict anaerobic conditions, easiness in infection of infectious microbe and the like of the flat plate method, the development of a rapid and accurate caproic acid bacteria quantitative detection technology is particularly urgent. The technology can greatly shorten the detection time, improve the monitoring efficiency, meet the requirements of dynamic real-time mastering of flora in the brewing fermentation and wastewater treatment processes, and provide basis for realizing accurate regulation. For example, in the maintenance of pit mud or the construction of artificial pit mud, accurate acquisition of the quantity of caproic acid bacteria can guide a bacteria liquid supplementing strategy to strengthen pit mud functions, in the treatment of wastewater, the treatment process and parameters can be optimized based on bacteria concentration data, the caproic acid synthesis efficiency can be improved, the operation cost can be reduced, and in the production of microbial inoculum, the culture conditions and process parameters can be optimized based on the caproic acid bacteria concentration data to obtain microbial inoculum products with higher concentration. Among various rapid detection means, qPCR technology is particularly suitable for detecting microorganisms difficult to culture such as caproic acid bacteria because of the advantages of real quantification, high sensitivity, strong specificity, rapid detection (< 2 hours), good repeatability and the like. Target genes commonly used in this technology include the conserved 16S rRNA gene, species-specific genes, and functional genes. However, the copy number of the 16S rRNA gene is different in genomes of different bacteria, and is usually 3-6, so that the quantitative result is seriously distorted and cannot reflect the actual cell number. And the conservation of the 16S rRNA gene is too high, so that it is difficult to design a specific primer capable of distinguishing closely related species or strains. Therefore, if a new accurate and quick detection method for caproic acid bacteria can be provided, the method has important application prospect in the fields of white spirit brewing and the like. Disclosure of Invention The invention aims to provide a quantitative detection method of caproic acid bacteria. In order to achieve the aim of the invention, the technical scheme adopted by the invention is that the primer pair comprises an upstream primer P4f, a downstream primer P4r and a downstream primer P4r, wherein the sequence of the upstream primer P4f is shown as SEQ ID NO. 8, and the sequence of the downstream primer P4r is shown as SEQ ID NO. 9. Correspondingly, a detection reagent, test paper or kit containing the primer pair. Correspondingly, the primer pair or the detection reagent, test paper or kit is applied to detection of caproic acid bacteria. Correspondingly, a method for detecting caproic acid bacteria is characterized in that qPCR specific primer pairs are designed based on recA genes of caproic acid bacteria to be detected, and the qPCR specific primer pairs are utilized to detect caproic acid bacteria. The primer pair comprises an upstream primer P4