CN-121975963-A - Primer probe combination for detecting bifidobacterium longum strain and subspecies and application thereof

CN121975963ACN 121975963 ACN121975963 ACN 121975963ACN-121975963-A

Abstract

The invention discloses a primer probe combination for detecting bifidobacterium longum strains and subspecies and application thereof, belonging to the technical field of molecular biology detection. The primer probe combination provided by the invention comprises a primer probe combination for detecting bifidobacterium longum, a primer probe combination for detecting bifidobacterium longum subspecies longum and a primer probe combination for detecting bifidobacterium subspecies longum infants. Based on the primer probe combination, the invention respectively develops a single PCR detection method and a triple PCR detection method for detecting bifidobacterium longum strains and subspecies. Experimental results show that the detection method provided by the invention can accurately detect the bifidobacterium longum at the seed level and can accurately detect the bifidobacterium longum subspecies and the bifidobacterium longum infant subspecies at the subspecies level. The invention lays a foundation for strain resource development at the subspecies level of bifidobacterium longum and authenticity identification of products containing bifidobacterium longum, and provides a more convenient method for detecting bifidobacterium longum strains and subspecies.

Inventors

WANG QI
WEI HAIYAN
XU LEIRUI
LI DAN
ZHANG XIAOLONG
WANG WEI
WEI YONGXIN
ZHAO XIAOJUAN

Assignees

中国海关科学技术研究中心
国家食品安全风险评估中心

Dates

Publication Date: 20260505
Application Date: 20260227

Claims (10)

1. A primer probe combination for detecting bifidobacterium longum strains and/or subspecies, which is characterized by comprising a primer probe group for detecting bifidobacterium longum, a primer probe group for detecting bifidobacterium longum subspecies and a primer probe group for detecting bifidobacterium longum subspecies; The primer probe group for detecting the bifidobacterium longum consists of an upstream primer with a nucleotide sequence shown as SEQ ID NO.1, a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3; The primer probe group for detecting the bifidobacterium longum subspecies longum consists of an upstream primer with a nucleotide sequence shown as SEQ ID NO.5, a downstream primer with a nucleotide sequence shown as SEQ ID NO.6 and a probe with a nucleotide sequence shown as SEQ ID NO. 7; the primer probe group for detecting the bifidobacterium longum subspecies infantis consists of an upstream primer with a nucleotide sequence shown as SEQ ID NO.9, a downstream primer with a nucleotide sequence shown as SEQ ID NO.10 and a probe with a nucleotide sequence shown as SEQ ID NO. 11.
2. Use of the primer probe combination of claim 1 in any of the following: (1) Application in preparing a single detection kit for detecting bifidobacterium longum strains; (2) Application in preparing a single detection kit for detecting bifidobacterium longum subspecies; wherein the bifidobacterium longum subspecies are bifidobacterium subspecies longum or bifidobacterium subspecies longum infantis.
3. A single detection kit for detecting bifidobacterium longum species and/or subspecies, characterized in that the single detection kit comprises the primer probe combination of claim 1.
4. A method for the single-load detection of bifidobacterium longum, bifidobacterium longum subspecies longum and bifidobacterium subspecies longum infancy for non-diagnostic purposes, comprising the steps of: Extracting genome DNA of a sample to be detected, and carrying out fluorescent PCR detection by using the single detection kit according to claim 3 by taking the genome DNA as a template; If the sample to be detected is detected by using the primer probe group of the bifidobacterium longum, the amplification curve exists, and the Ct value is less than or equal to 25, the bifidobacterium longum exists in the sample to be detected; If the sample to be detected is detected by using the primer probe combination of the long bifidobacterium subspecies, the amplification curve is available, and the Ct value is less than or equal to 25, judging that the long bifidobacterium subspecies are not detected in the sample to be detected; If the sample to be detected is detected by using the primer probe combination of the bifidobacterium longum subspecies infancy, the amplification curve is provided, and the Ct value is less than or equal to 25, judging that the bifidobacterium longum subspecies infancy exists in the sample to be detected; if the sample to be detected has an amplification curve, but the Ct value is less than 30 and 25< 25 >, the sample is determined to be an uncertain sample, and the sample extraction amount is required to be increased for retesting.
5. The method according to claim 4, wherein the reaction system for fluorescent PCR detection is 2 XPCR Mix 10. Mu.L, 1. Mu.L each of the upstream and downstream primers, 0.5. Mu.L each of the probe, 1. Mu.L each of the DNA template, and ddH 2 O to 20. Mu.L; The reaction conditions of the fluorescent PCR detection are 95 ℃ 10 min hot start, 95 ℃ denaturation 10s, 60 ℃ annealing and extension 30 s, and the total period is 35 cycles.
6. Use of the primer probe combination of claim 1 in any of the following: (1) The application of the kit in preparing a triple detection kit for detecting bifidobacterium longum strains; (2) The application of the kit in preparing a triple detection kit for detecting bifidobacterium longum subspecies; wherein the bifidobacterium longum subspecies are bifidobacterium subspecies longum or bifidobacterium subspecies longum infantis; The probe for detecting the bifidobacterium longum is added with a Cy5 fluorescent group, the probe for detecting the bifidobacterium longum subspecies longum is added with a VIC fluorescent group, and the probe for detecting the bifidobacterium longum subspecies infancy is added with a FAM fluorescent group.
7. A triple detection kit for detecting bifidobacterium longum species and/or subspecies, characterized in that the triple detection kit comprises the primer probe combination of claim 1; the bifidobacterium longum subspecies are bifidobacterium subspecies longum or bifidobacterium longum subspecies infantis; The probe for detecting the bifidobacterium longum is added with a Cy5 fluorescent group, the probe for detecting the bifidobacterium longum subspecies longum is added with a VIC fluorescent group, and the probe for detecting the bifidobacterium longum subspecies infancy is added with a FAM fluorescent group.
8. A method for the triple detection of bifidobacterium longum, bifidobacterium longum subspecies longum and bifidobacterium subspecies longum infantis for non-diagnostic purposes, comprising the steps of: Extracting genome DNA of a sample to be detected, and performing fluorescence PCR detection by using the triple detection kit according to claim 7 and taking the genome DNA as a template; if the sample to be detected has an amplification curve in a Cy5 detection channel and the Ct value is less than or equal to 25, judging that bifidobacterium longum exists in the sample to be detected; If the sample to be detected has an amplification curve in a VIC detection channel and the Ct value is less than or equal to 25, judging that bifidobacterium longum subspecies are present in the sample to be detected; if the sample to be detected has an amplification curve in the FAM detection channel and the Ct value is less than or equal to 25, judging that bifidobacterium longum subspecies infancy exist in the sample to be detected; If the sample to be detected has an amplification curve in the corresponding fluorescence detection channel, but the Ct value is less than 30 and is 25< 25 >, the sample to be detected is determined to be an uncertain sample, and the sample extraction amount is required to be increased for retesting.
9. The method of claim 8, wherein the reaction system of the fluorescent PCR assay is 2 XPCR Mix 10. Mu.L, 1. Mu.L each of the upstream and downstream primers, 0.5. Mu.L each of the probes, 1. Mu.L of the DNA template, and ddH 2 O to 20. Mu.L; The reaction conditions of the fluorescent PCR detection are 95 ℃ 10 min hot start, 95 ℃ denaturation 10s, 60 ℃ annealing and extension 30 s, and the total period is 35 cycles.
10. Use of a single detection kit according to claim 3 or a triple detection kit according to claim 7 for the authenticity of a product containing bifidobacterium longum subspecies, wherein said bifidobacterium longum subspecies comprise bifidobacterium longum subspecies and bifidobacterium longum subspecies infantis.

Description

Primer probe combination for detecting bifidobacterium longum strain and subspecies and application thereof Technical Field The invention relates to the field of molecular biology detection, in particular to a primer probe combination for detecting bifidobacterium longum strains and subspecies and application thereof. Background The bifidobacterium of the bifidobacterium longum is one of core flora for maintaining the microecology balance of intestinal tracts of human beings and animals, has the probiotic functions of regulating the balance of the intestinal flora, enhancing the immunity, reducing the cholesterol and the like, and is a probiotic beneficial to human bodies. Bifidobacterium longum was initially divided into 3 subspecies, namely Bifidobacterium longum subspecies Bifidobacterium longum subsp. Longum, bifidobacterium longum subsp. Infantis (Bifidobacterium longum subsp. Infantis) and Bifidobacterium subsp. Suis (Bifidobacterium longum subsp. Suis). The long bifidobacterium subspecies are firstly separated from the intestinal tracts of adults, the long bifidobacterium subspecies are one of dominant bacterial groups in the intestinal tracts of infants, and are closely related to the healthy development of infants, and the long bifidobacterium subspecies are important components of the microbial bacterial groups in the intestinal tracts of pigs. The bifidobacterium longum subspecies suilum (Bifidobacterium longum subsp. Suilum) were first described and named in 2015 and were based on a more refined genotyping study of a group isolated from Bifidobacterium longum subsp. Suis that was unable to produce urease. These 4 subspecies differ somewhat in biological properties, ecological distribution and function. In practical application, only bifidobacterium longum subspecies and bifidobacterium longum subspecies are formally listed in a bacterial list for food, and the bifidobacterium longum subspecies can be applied to foods such as health care products, probiotic preparations, fermented dairy products, probiotic beverages, milk powder and the like. The method has the advantages of accurately detecting and distinguishing the bifidobacterium longum subspecies and the bifidobacterium longum subspecies, and has important significance in the aspects of researching the structure of intestinal microbial communities, evaluating the quality and efficacy of probiotic products, developing the control of related diseases, developing probiotic strain resources and the like. In the field of foods, specific subspecies of bifidobacterium longum contained in products are clarified, the consistency of product quality and efficacy is guaranteed, and corresponding microecological preparations are developed aiming at the characteristics of different subspecies in the research and development of medicines, so that the treatment effect can be improved. However, the traditional detection method, such as morphological observation, physiological and biochemical identification, is complex in operation, long in time consumption and low in accuracy. The conventional 16S rRNA strain identification method cannot realize subspecies discrimination. At present, methods such as whole genome sequencing, phylogenetic tree, PCR-RFLP, PCR-DGGE and the like are mostly used for distinguishing different subspecies, but the methods are complex in operation, long in time consumption and high in cost. Therefore, the development of the fluorescence PCR method which can accurately, quickly and efficiently detect the bifidobacterium longum, the bifidobacterium longum subspecies longum and the bifidobacterium infantis subspecies longum in a single detection mode and multiple detection modes has important practical significance. Disclosure of Invention The invention aims to provide a primer probe combination for detecting bifidobacterium longum strains and subspecies and application thereof, so as to solve the problems in the prior art, and provides a primer probe combination for detecting bifidobacterium longum strains and subspecies, which can be used for single PCR detection and triple PCR detection, can be used for accurately detecting bifidobacterium longum at a seed level and accurately detecting bifidobacterium subspecies and bifidobacterium subspecies infantis at a subspecies level, and can realize authenticity identification of products containing bifidobacterium longum. The invention provides a more convenient method for detecting bifidobacterium longum strains and subspecies. In order to achieve the above object, the present invention provides the following solutions: The invention provides a primer probe combination for detecting bifidobacterium longum strains and/or subspecies, which comprises a primer probe group for detecting bifidobacterium longum, a primer probe group for detecting bifidobacterium longum subspecies and a primer probe group for detecting bifidobacterium longum subspecies; The primer probe group for detecting the bifido