CN113718047B

CN113718047B - Kit for detecting 10 bacteria in human breast milk by fluorescence quantitative method and application thereof

Info

Publication number: CN113718047B
Application number: CN202111296619.8A
Authority: CN
Inventors: 郑明权; 李巧玲
Original assignee: Edvance Beijing Medical Laboratory Co ltd
Current assignee: Edvance Beijing Medical Laboratory Co ltd
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2022-02-18
Anticipated expiration: 2041-11-04
Also published as: CN113718047A

Abstract

The invention discloses a kit for detecting 10 bacteria in human breast milk by a fluorescence quantitative method and application thereof. The kit comprises a multiplex PCR primer probe composition for identifying or assisting in identifying bacteria in breast milk, wherein the bacteria are bacteria in 10 genera or less from the 10 genera consisting of bifidobacterium, acinetobacter, corynebacterium, streptococcus, staphylococcus, morganella, providencia, bacillus, stenotrophomonas and aquaticus, and the multiplex PCR primer probe composition comprises single-stranded DNA with a nucleotide sequence from sequence 2 to sequence 31 in a sequence table. The invention can monitor the detection process in real time, can restore the original flora state of the sample, has accurate and rapid detection method, can simultaneously carry out qualitative detection and quantitative detection on 10 species in human breast milk, can also carry out single-species detection or combined detection on multiple species, and can carry out combined detection on several species according to fluorescent markers at will.

Description

Kit for detecting 10 bacteria in human breast milk by fluorescence quantitative method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting 10 bacteria in human breast milk by a fluorescence quantitative method and application thereof.

Background

In recent years, secondary sequencing studies have shown that there are nearly thousands of bacteria in breast milk, and microorganisms in human milk that have been isolated and identified are mainly staphylococcus, streptococcus, enterococcus, lactobacillus, bifidobacterium, leuconostoc, and the like. There is increasing evidence that microorganisms contained in human milk originate from pathways other than environmental pollution, i.e., bacteria in the maternal intestine migrate through the intrinsic pathway to the mammary gland (gut-mammary pathway) and then through lactation into the infant. Meanwhile, related bacteria in breast milk are the first colonizing bacteria in the intestinal tract of the infant, and help the infant to establish an intestinal symbiotic bacteria system.

The influence of microorganisms in breast milk on the health of mothers and infants is of great concern. Breast feeding is helpful for the growth and colonization of intestinal probiotic flora of infants, and certain conditional microorganisms in breast milk play a role in the activation process of the endogenous immune system of a newborn, programming the immune system of the newborn, the development and maturation of intestinal immune function and reducing the disease susceptibility track. Meanwhile, the conditioned microorganisms are also related to the occurrence risk and treatment effect of mastitis, and influence the risk of long-term chronic diseases of mothers and infants.

Research has specifically investigated the composition of the mother's milk flora of lactating mothers living in cities in china, and it has been found that streptococci, acinetobacter and staphylococci are dominant genera whether using standard non-sterile sampling or sterile sampling. The composition of the human milk flora may be influenced by many factors including genetic factors, maternal health and nutritional status, mode of delivery, lactation, and geographic location.

At present, the detection method of microorganisms in human milk is basically 16S second generation sequencing technology. The 16S rRNA gene is a DNA sequence corresponding to coding rRNA on bacteria, exists in genomes of all bacteria, generally consists of a conserved region and a hypervariable region, the conserved region has no obvious difference among the bacteria and can be used for constructing a unified evolutionary tree of all life, and the hypervariable region has certain difference among different bacteria and can accurately identify floras to taxonomic genus or even species level. 16S rRNA gene sequencing is to select one or several hypervariable regions, utilize the conserved region to design universal primer for PCR amplification, and then perform sequencing analysis and strain identification on the hypervariable regions to obtain the diversity of microbial community in breast milk. The second-generation sequencing has the advantages of low cost and easy operation in large-sample sequencing, can obtain all microorganism information in a sample, and can restore the bacterial species and the bacterial abundance distribution condition of a microecosystem in breast milk to a certain extent. Meanwhile, the second-generation sequencing also has the defect of longer period when sequencing small samples: because the library building and sequencing process needs to be performed through multiple PCR amplifications, the sequencing result can not effectively restore the original information of the sample to a certain extent due to the deviation in the PCR amplifications.

Disclosure of Invention

The problems in the existing second generation sequencing technology are as follows: sequencing cycles for small sample sizes are long.

To solve the above problems, the present invention provides, in a first aspect, a multiplex PCR primer probe composition for identifying or assisting in identifying bacteria in breast milk, the bacteria being bacteria of 10 or less of the 10 genera consisting of Bifidobacterium, Acinetobacter, Corynebacterium, Streptococcus, Staphylococcus, Morganella, providencia, Bacillus, stenotrophomonas and Aquifex, the multiplex PCR primer probe composition comprising 1-FRP, 2-FRP, 3-FRP, 5-FRP, 6-FRP, 7-FRP, 8-FRP, 9-FRP, 10-FRP and 11-FRP;

the 1-FRP is a specific primer and a Probe of the acinetobacter, and consists of a primer 1-FP, a primer 1-RP and a Probe 1-Probe, wherein the primer 1-FP is a single-stranded DNA shown in SEQ ID No.2, the primer 1-RP is a single-stranded DNA shown in SEQ ID No.3, and the Probe 1-Probe is a single-stranded DNA shown in SEQ ID No. 4; the 2-FRP is a specific primer and a Probe of the genus Bifidobacterium, and consists of a primer 2-FP, a primer 2-RP and a Probe 2-Probe, wherein the primer 2-FP is a single-stranded DNA shown in SEQ ID No.5, the primer 2-RP is a single-stranded DNA shown in SEQ ID No.6, and the Probe 2-Probe is a single-stranded DNA shown in SEQ ID No. 7; the 3-FRP is a specific primer and a Probe of corynebacterium, and consists of a primer 3-FP, a primer 3-RP and a Probe 3-Probe, wherein the primer 3-FP is a single-stranded DNA shown in SEQ ID No.8, the primer 3-RP is a single-stranded DNA shown in SEQ ID No.9, and the Probe 3-Probe is a single-stranded DNA shown in SEQ ID No. 10; the 5-FRP is a specific primer and a Probe of stenotrophomonas, and consists of a primer 5-FP, a primer 5-RP group and a Probe 5-Probe, wherein the primer 5-FP is single-stranded DNA shown in SEQ ID No.11, the primer 5-RP is single-stranded DNA shown in SEQ ID No.12, and the Probe 5-Probe is single-stranded DNA shown in SEQ ID No. 13; the 6-FRP is a specific primer and a Probe of streptococcus and consists of a primer 6-FP, a primer 6-RP and a Probe 6-Probe, wherein the primer 6-FP is a single-stranded DNA shown in SEQ ID No.14, the primer 6-RP is a single-stranded DNA shown in SEQ ID No.15, and the Probe 6-Probe is a single-stranded DNA shown in SEQ ID No. 16; the 7-FRP is a specific primer and a Probe of bacillus, and consists of a primer 7-FP, a primer 7-RP and a Probe 7-Probe, wherein the primer 7-FP is single-stranded DNA shown in SEQ ID No.17, the primer 7-RP is single-stranded DNA shown in SEQ ID No.18, and the 7-Probe is single-stranded DNA shown in SEQ ID No. 19; the 8-FRP is a specific primer and a Probe of the genus waterborne, and consists of a primer 8-FP, a primer 8-RP and a Probe 8-Probe, wherein the primer 8-FP is a single-stranded DNA shown in SEQ ID No.20, the primer 8-RP is a single-stranded DNA shown in SEQ ID No.21, and the Probe 8-Probe is a single-stranded DNA shown in SEQ ID No. 22; the 9-FRP is a specific primer and a Probe of Morganella, and consists of a primer 9-FP, a primer 9-RP and a Probe 9-Probe, wherein the primer 9-FP is a single-stranded DNA shown in SEQ ID No.23, the primer 9-RP is a single-stranded DNA shown in SEQ ID No.24, and the Probe 9-Probe is a single-stranded DNA shown in SEQ ID No. 25; the 10-FRP is a specific primer and a Probe of providencia, and consists of a primer 10-FP, a primer 10-RP and a Probe 10-Probe, wherein the primer 10-FP is single-stranded DNA shown in SEQ ID No.26, the primer 10-RP is single-stranded DNA shown in SEQ ID No.27, and the Probe 10-Probe is single-stranded DNA shown in SEQ ID No. 28; the 11-FRP is a specific primer and a Probe of staphylococcus, and consists of a primer 11-FP, a primer 11-RP and a Probe 11-Probe, wherein the primer 11-FP is single-stranded DNA shown in SEQ ID No.29, the primer 11-RP is single-stranded DNA shown in SEQ ID No.30, and the Probe 11-Probe is single-stranded DNA shown in SEQ ID No. 31.

Further, in the above-mentioned multiplex PCR primer Probe composition, the primer 1-FP, the primer 1-RP, the Probe 1-Probe, the primer 2-FP, the primer 2-RP, the Probe 2-Probe, the primer 3-FP, the primer 3-RP, the Probe 3-Probe, the primer 5-FP, the primer 5-RP, the Probe 5-Probe, the primer 6-FP, the primer 6-RP, the Probe 6-Probe, the primer 7-FP, the primer 7-RP, the Probe 7-Probe, the primer 8-FP, the primer 8-RP, the Probe 8-Probe, the primer 9-FP, the primer 9-RP, the Probe 9-Probe, the Probe 3-Probe, the Probe 5-Probe, the primer 5-FP, the primer 6-FP, the Probe 6-Probe, the primer 7-Probe, the Probe 8-FP, the primer 8-RP, the Probe 8-Probe, the Probe 9-FP, the Probe 9-Probe, The amounts of the primer 10-FP, the primer 10-RP, the Probe 10-Probe, the primer 11-FP, the primer 11-RP and the Probe 11-Probe are the same.

Further, in the multiplex PCR primer Probe composition, the composition further comprises an internal reference-FRP, wherein the internal reference-FRP is composed of a primer internal reference-FP, a primer internal reference-RP and a Probe internal reference-Probe, the primer internal reference-FP is single-stranded DNA shown in SEQ ID No.32, the primer internal reference-RP is single-stranded DNA shown in SEQ ID No.33, and the Probe internal reference-Probe is single-stranded DNA shown in SEQ ID No. 34.

Further, in the multiplex PCR primer Probe composition, the amounts of the substances of the primer internal reference-FP, the primer internal reference-RP and the Probe internal reference-Probe are the same as the amount of the substance of the primer 1-FP.

In the above multiplex PCR primer probe composition, the 1-FRP, the 2-FRP and the 3-FRP are packaged together (this package is abbreviated as FRP-Mix 1). The 5-FRP, the 6-FRP, the 7-FRP and the 8-FRP are packaged together (the package is abbreviated as FRP-Mix 2). The 9-FRP, the 10-FRP, the 11-FRP and the internal reference-FRP are packaged together (the package is abbreviated as FRP-Mix 3).

In the multiplex PCR primer probe composition, the multiplex PCR may be: the 1-FRP, the 2-FRP, and the 3-FRP are used to perform triple PCR (i.e., the 1-FRP, the 2-FRP, and the 3-FRP are PCR in one system), the 5-FRP, the 6-FRP, the 7-FRP, and the 8-FRP are used to perform quadruple PCR (i.e., the 5-FRP, the 6-FRP, the 7-FRP, and the 8-FRP are PCR in one system), and the 9-FRP, 10-FRP, 11-FRP, and internal reference-FRP are used to perform quadruple PCR (i.e., the 9-FRP, 10-FRP, 11-FRP, and internal reference-FRP are PCR in one system).

In a second aspect, the present invention provides a reagent or kit for identifying or aiding in the identification of bacteria in the mother's milk, said reagent or kit comprising the multiplex PCR primer probe composition described above.

Further, the reagent or the kit further comprises a positive standard substance, wherein the positive standard substance can be a vector containing a DNA fragment with the nucleotide sequence shown in the 4287-position 5862 of SEQ ID No. 1.

In one embodiment of the invention, the positive standard is a plasmid with the nucleotide sequence of SEQ ID No. 1.

In a third aspect, the present invention provides the use of the multiplex PCR primer probe composition described above for the preparation of a reagent or kit for identifying or assisting in the identification of bacterial products (reagents or kits) in breast milk.

In a fourth aspect, the invention provides the use of the multiplex PCR primer probe composition as described above for identifying or assisting in identifying bacteria in breast milk.

In a fifth aspect, the invention provides the use of the reagent or the kit for identifying or assisting in identifying the microbial species in breast milk as described above in identifying or assisting in identifying bacteria in breast milk.

In a sixth aspect, the present invention also provides a method for identifying bacteria in breast milk, the method includes using genomic DNA of breast milk to be tested as a template, performing real-time fluorescence quantitative PCR detection with the above multiplex PCR primer probe composition or the above reagent or kit, and determining which genus or genera of bacteria in the above 10 genera of bacteria the breast milk to be tested contains.

Herein, the bacteria in the breast milk are bacteria of the genus 10, any 9, any 8, any 7, any 6, any 5, any 4, any 3, any 2 or any 1 of the following 10 genera: bifidobacterium (Bifidobacterium) Acinetobacter (A), (B) and (C)Acinetobacter) Corynebacterium genus (A), (B), (C)Corynebacterium) Streptococcus (C.A.)Streptococcus) Staphylococcus genus (1)Staphylococcus) Morganella genus (A), (B), (C), (B), (C)Morganella) Provedasius genus (A), (B), (C)Providencia) Bacillus (B) and (C)Bacillus) Stenotrophomonas (II), (III)Stenotrophomonas) And genus Hizikia (Enhydrobacter）。

The method provided by the invention has the following advantages: firstly, when the sample to be detected is small in sample amount, the method is simple and convenient, and the period is fast; secondly, the real-time fluorescent quantitative PCR (qRT-PCR) of the probe method can monitor the detection process in real time and reduce the original flora state of the sample; high targeting performance; single species detection can be carried out; or combined detection of multiple genera, and combined detection of several species according to fluorescent markers in any combination; and fourthly, species qualitative detection and quantitative detection can be realized simultaneously, and the quantification is more accurate. The detection method provided by the invention is accurate and rapid, can be used for simultaneously carrying out qualitative detection and quantitative detection on 10 species in human breast milk, and has guiding significance on infant probiotic supplementation according to the detection result.

Drawings

FIG. 1 is a diagram showing information on 10 genus bacteria detected by the multiplex PCR primer probe composition provided in the present invention.

FIG. 2 shows the nucleotide sequence of the multiplex PCR primer probe composition and the fluorescence labeling information of the probe.

FIG. 3 is a graph of the first part of the data for sensitivity detection of multiplex PCR primer probe compositions.

FIG. 4 is a second set of data for sensitivity detection of multiplex PCR primer probe compositions.

FIG. 5 is standard curve information for multiplex PCR primer probe compositions.

FIG. 6 is a graph of multiplex PCR primer probe compositions for determining the number of amplification cycles of bacteria in 10 breast milk samples.

FIG. 7 shows the results of multiplex PCR primer probe compositions for the determination of bacteria in 3 breast milk samples, negative controls and positive standard plasmids.

FIG. 8 shows the detection data of 3 cases of bacteria in breast milk samples in the second generation sequencing assay.

FIG. 9 is the species abundance of bacteria in 3 breast milk samples determined by second generation sequencing.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

According to the invention, 10 characteristic genera of bacteria in breast milk are screened out through 500 examples of matching biological information analysis of the breast milk 16SrDNA second-generation sequencing data owned by an Edwardsies medical examination laboratory, and specific primers and probes and primers and probes of endogenous references are designed aiming at the 10 genera to design and construct a positive standard substance. The matched biological information analysis method comprises the following specific steps:

step 1, performing data quality control by using fastp software based on a high throughput sequencing (NGS) original sequence, wherein the fastp downloads https:// github.com/OpenGene/fastp;

step 2. use the qiime2 analysis software for ASV (amplicon sequence variant) analysis and species annotation (SILVA database), qiime2 official website https:// qiime2. org;

step 3. filtering the annotated ASV to cyanobacteria/mitochondria/chloroplasts etc. and ASV with total reads less than 20 in all samples;

and 4, selecting ASV with average abundance of not less than 0.5% in 95% of samples as core ASV, and taking the corresponding bacteria as a final screening and identifying result.

The characteristic genera of 10 bacteria are screened out by biological information analysis, wherein: the specific information of the genus probiotic 1, the genus of conditional pathogenic bacteria 7 and the genus of neutrophilic bacteria 3 and the numbers of corresponding primers and probes are shown in figure 1. The nucleotide sequences of specific primers and probes designed for bacteria of genus 10, and primers and probes of endogenous reference are shown in FIG. 2.

Construction of positive standard quality particles: according to the detection areas of the primer and the probe set, a positive standard sample is designed, the total length is 1576 nucleotides, and the nucleotide sequence is shown as 4287-5862 th site of SEQ ID No. 1. And (4) constructing a positive standard substance sequence into a pUC57 vector by Shanghai workers to obtain a positive standard substance recombinant plasmid pUC 57-positive. The nucleotide sequence of pUC57-positive is shown as SEO ID No.1, wherein the 1 st-4286 th position of SEO ID No.1 is the nucleotide sequence of pUC57 of the framework vector, and the 4286 th-5862 th position of SEQ ID No.1 is the positive standard substance sequence.

Example 1 specificity verification experiment of Positive Standard and primer Probe composition

1.1. Verification method

The specificity verification of the positive standard substance and primer probe composition comprises the following steps:

1) 2 μ L of the extract is taken to have a concentration of 2.0X 10⁴Taking copy/mu L of positive standard quality particle pUC57-positive solution as a template for specific detection;

2) the primer and probe sets were divided into 3 groups based on the difference of the fluorophores (FIG. 2), and the three primer probe set solutions were: FRP-Mix 1, FRP-Mix 2 and FRP-Mix 3.

The FRP-Mix 1 solution consists of 3 primer Probe compositions with the names of 1-FRP, 2-FRP and 3-FRP respectively and nuclease-free water, wherein the 1-FRP consists of a primer 1-FP, a primer 1-RP and a Probe 1-Probe, the 1-FP is single-stranded DNA shown in SEQ ID No.2, the 1-RP is single-stranded DNA shown in SEQ ID No.3, the nucleotide sequence of the 1-Probe is shown in SEQ ID No.4, the 5 'end of the 1-Probe is connected with a fluorescent group 6-FAM, and the 3' end is connected with a quenching group BHQ 1; the 2-FRP consists of a primer 2-FP, a primer 2-RP and a Probe 2-Probe, wherein the 2-FP is single-stranded DNA shown in SEQ ID No.5, the 2-RP is single-stranded DNA shown in SEQ ID No.6, the nucleotide sequence of the 2-Probe is shown in SEQ ID No.7, the 5 'end of the 2-Probe is connected with a fluorescent group HEX, and the 3' end of the 2-Probe is connected with a quenching group BHQ 1; the 3-FRP consists of a primer 3-FP, a primer 3-RP and a Probe 3-Probe, wherein the 3-FP is single-stranded DNA shown in SEQ ID No.8, the 3-RP is single-stranded DNA shown in SEQ ID No.9, the nucleotide sequence of the 3-Probe is shown in SEQ ID No.10, the 5 'end of the 3-Probe is connected with a fluorescent group ROX, and the 3' end is connected with a quenching group BHQ 2.

The FRP-Mix 2 solution comprises 4 primer Probe compositions and nuclease-free water, wherein the names of the 4 primer Probe compositions are 5-FRP, 6-FRP, 7-FRP and 8-FRP respectively, the 5-FRP comprises a primer 5-FP, a primer 5-RP and a Probe 5-Probe, the 5-FP is single-stranded DNA shown in SEQ ID No.11, the 5-RP is single-stranded DNA shown in SEQ ID No.12, the nucleotide sequence of the 5-Probe is shown in SEQ ID No.13, the 5 'end of the 5-Probe is connected with a fluorescent group 6-FAM, and the 3' end of the 5-Probe is connected with a quenching group BHQ 1; the 6-FRP consists of a primer 6-FP, a primer 6-RP and a Probe 6-Probe, wherein the 6-FP is single-stranded DNA shown in SEQ ID No.14, the 6-RP is single-stranded DNA shown in SEQ ID No.15, the nucleotide sequence of the 6-Probe is shown in SEQ ID No.16, the 5 'end of the 6-Probe is connected with a fluorescent group HEX, and the 3' end of the 6-Probe is connected with a quenching group BHQ 1; the 7-FRP consists of a primer 7-FP, a primer 7-RP and a Probe 7-Probe, wherein the 7-FP is a single-stranded DNA shown in SEQ ID No.17, the 7-RP is a single-stranded DNA shown in SEQ ID No.18, the nucleotide sequence of the 7-Probe is shown in SEQ ID No.19, the 5 'end of the 7-Probe is connected with a fluorescent group ROX, and the 3' end of the 7-Probe is connected with a quenching group BHQ 2; the 8-FRP consists of a primer 8-FP, a primer 8-RP and a Probe 8-Probe, wherein the 8-FP is single-stranded DNA shown in SEQ ID No.20, the 8-RP is single-stranded DNA shown in SEQ ID No.21, the nucleotide sequence of the 8-Probe is shown in SEQ ID No.22, the 5 'end of the 6-Probe is connected with a fluorescent group Cy5, and the 3' end of the 6-Probe is connected with a quenching group BHQ 2.

The FRP-Mix3 solution comprises 4 primer Probe compositions and nuclease-free water with the names of 9-FRP, 10-FRP, 11-FRP and internal reference-FRP respectively, wherein the 9-FRP comprises a primer 9-FP, a primer 9-RP and a Probe 9-Probe, the 9-FP is single-stranded DNA shown in SEQ ID No.23, the 9-RP is single-stranded DNA shown in SEQ ID No.24, the nucleotide sequence of the 9-Probe is shown in SEQ ID No.25, the 5 'end of the 9-Probe is connected with a fluorescent group 6-FAM, and the 3' end is connected with a quenching group BHQ 1; the 10-FRP consists of a primer 10-FP, a primer 10-RP and a Probe 10-Probe, wherein the 10-FP is a single-stranded DNA shown in SEQ ID No.26, the 10-RP is a single-stranded DNA shown in SEQ ID No.27, the nucleotide sequence of the 10-Probe is shown in SEQ ID No.28, the 5 'end of the 10-Probe is connected with a fluorescent group HEX, and the 3' end of the 10-Probe is connected with a quenching group BHQ 1; the 11-FRP consists of a primer 11-FP, a primer 11-RP and a Probe 11-Probe, wherein the 11-FP is a single-stranded DNA shown in SEQ ID No.29, the 11-RP is a single-stranded DNA shown in SEQ ID No.30, the nucleotide sequence of the 11-Probe is shown in SEQ ID No.31, the 5 'end of the 11-Probe is connected with a fluorescent group ROX, and the 3' end of the 11-Probe is connected with a quenching group BHQ 2; the internal reference-FRP consists of a primer internal reference-FP, a primer internal reference-RP and a Probe internal reference-Probe, wherein the internal reference-FP is single-stranded DNA shown in SEQ ID No.32, the internal reference-RP is single-stranded DNA shown in SEQ ID No.33, the nucleotide sequence of the internal reference-Probe is shown in SEQ ID No.34, the 5 'end of the internal reference-Probe is connected with a fluorescent group Cy5, and the 3' end of the internal reference-Probe is connected with a quenching group BHQ 2.

In FRP-Mix 1, the contents of primer 1-FP, primer 1-RP, Probe 1-Probe, primer 2-FP, primer 2-RP, Probe 3-Probe, primer 3-FP, primer 3-RP and Probe 3-Probe are all 0.5. mu.M. In FRP-Mix 2, the contents of primer 5-FP, primer 5-RP, Probe 5-Probe, primer 6-FP, primer 6-RP, Probe 6-Probe, primer 7-FP, primer 7-RP, Probe 7-Probe, primer 8-FP, primer 8-RP and Probe 8-Probe are all 0.5. mu.M. In the FRP-Mix3, the contents of a primer 9-FP, a primer 9-RP, a Probe 9-Probe, a primer 10-FP, a primer 10-RP, a Probe 10-Probe, a primer 11-FP, a primer 11-RP, a Probe 11-Probe, a primer internal reference-FP, a primer internal reference-RP and a Probe internal reference-Probe are all 0.5 mu M. If 10 species are to be detected in a single sample, 3 tubes of detection reaction (containing one internal reference gene) are needed.

3) 2 mu.L of positive standard quality particle pUC57-positive solution was used as a template to prepare 3 reaction systems, namely, FRP-Mix 1 system, FRP-Mix 2 system and FRP-Mix3 system.

The FRP-Mix 1 system, the FRP-Mix 2 system and the FRP-Mix3 system are different only in primer probe set solution, and the primer probe set solution of the FRP-Mix 1 system, the FRP-Mix 2 system and the FRP-Mix3 system is FRP-Mix 1 solution, FRP-Mix 2 solution and FRP-Mix3 solution respectively. The FRP-Mix 1 system, the FRP-Mix 2 system and the FRP-Mix3 system all comprise the following components: 2 XProbe mix (Kangshiji, cat # CW 0932M) 10. mu.L, primer Probe set solution 5. mu.L, positive standard quality plasmid pUC57-positive solution 2. mu.L, enzyme-free nucleic acid water 3. mu.L, total 20. mu.L.

The FRP-Mix 1 system, the FRP-Mix 2 system and the FRP-Mix3 system are subjected to fluorescence quantitative detection according to the following reaction procedures: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15s, annealing/extension at 65 ℃ for 60s, 40 cycles, and fluorescence collection after the reaction is finished.

1.2. Detection results and result analysis

The baseline is taken as the threshold (threshold). Rn (normalized reporter) is the ratio of the fluorescence emission intensity of the fluorescent reporter group to the fluorescence emission intensity of the reference dye. Δ Rn is the normalized result obtained after baseline subtraction of Rn (Δ Rn = Rn-baseline), representing the amount of PCR amplification product. Ct (cycle threshold) refers to the number of cycles that the fluorescence signal in the reaction tube has undergone to reach a set threshold.

Ct values detected for positive standard quality plasmid pUC57-positive were as follows: ct value 15.87 for group 1, Ct value 16.01 for group 2, Ct value 15.42 for group 3, Ct value 15.87 for group 5, Ct value 17.03 for group 6, Ct value 15.7 for group 7, Ct value 15.05 for group 8, Ct value 15.74 for group 9, Ct value 15.75 for group 10, Ct value 16.20 for group 11, and Ct value 17.49 for internal reference.

The specificity experiment shows that the primer probe composition and the internal reference primer probe composition developed by the kit can effectively amplify a specific region of a positive standard plasmid pUC57-positive solution to-be-detected species, and have high specificity (Ct values are basically close to but not completely the same because of difference in amplification efficiency of the primers and the probes).

Example 2 sensitivity detection and Standard Curve establishment

2.1. Sensitive detection

The sensitivity of the primer and probe compositions provided in example 1 was tested using a positive standard quality plasmid pUC 57-positive. According to the conversion formula "(6.02X 1023) × (ng/. mu.l X10)^-9) /(plasmid length (bp) × 660) = copy number/. mu.l "a positive standard plasmid pUC57-positive was prepared with nuclease-free water into a solution having a concentration (copy number/. mu.L) of 1E 8. Taking the positive standard substance to dilute in a multiple ratio to obtain standard substance solutions with 7 concentration gradients (copy number/. mu.L): 1E2, 1E3, 1E4, 1E5, 1E6, 1E7, and 1E 8. Using the 7 concentration-gradient standards as amplification templates, 2. mu.L of each of the 7 concentration-gradient standards was used in the amplification reaction, and the reaction system and the reaction procedure were the same as in example 1. As shown in FIGS. 3 and 4, when the concentration of the positive standard substance (copy number/. mu.L) is 1E2, no corresponding fluorescent signal is detected in a part of the target genus (no fluorescent signal is detected as indicated by "-" in FIG. 3); therefore, in the preparation of the calibration curve, 6 consecutive concentrations of the measurement data 1E3, 1E4, 1E5, 1E6, 1E7 and 1E8 were taken.

2.2. Standard curve establishment

The concentrations (concentration unit is copy number/. mu.L) of the positive plasmids in 2.1 are respectively 1E8, 1E7, 1E6, 1E5, 1E4 and 1E3, the logarithm of the initial copy number of the standard (log (initial copy number of template DNA)) is used as an abscissa, and the Ct value is used as an ordinate to prepare a standard curve. The slope and Y-intercept of the detected bacteria and the standard curve corresponding to 10 primer probe sets and the R2 data are shown in fig. 5, and the experiment can establish the standard curves of 10 genus bacteria and 1 reference standard curve at most, and the correlation coefficient R2 is greater than 0.98.

Example 3 specificity verification experiment

The method of the present invention was validated using a commercial simulated microbial community (zymbiomistm, D6300). The composition of the simulated microbial community comprises eight bacterial strains and two fungal strains. It includes three gram-negative bacteria that are susceptible to lysis (e.g., E.coli), five gram-positive bacteria that are difficult to lyse (e.g., Listeria monocytogenes), and two yeasts that are difficult to lyse (e.g., Cryptococcus neoformans). The simulated microbial community contains microorganisms such as Pseudomonas aeruginosa (Pseudomonas aeruginosa), Escherichia coli (Escherichia coli), Salmonella enterica, Lactobacillus fermentum (Lactobacillus fermentum), Enterococcus faecalis (Enterococcus faecalis), Staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Bacillus subtilis (Bacillus subtilis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Cryptococcus neoformans (Cryptococcus neoformas), wherein seven strains are known human pathogens and have been completely inactivated by DNA/RNA Shield.

The staphylococcus aureus and the bacillus subtilis in the simulated microbial community belong to the detectable species of the primer probe composition provided by the invention. The remaining 8 species do not belong to the detectable species of the primer probe composition of the invention.

The detection method comprises the following steps: genomic DNA of a simulated microbial community (ZymBIOMICSTM, D6300) was extracted, and the concentration of the detected nucleic acid was 9.1 ng/. mu.L. Respectively using the primer probe sets FRP-Mix 1, FRP-Mix 2 and FRP-Mix3 for detection, and taking 2 mu L of each detection hole as a template for detection; the procedure was as in example 1.

The specific detection result is as follows: the Ct value of the 7 th group was 17.6 at a reduced concentration of 5.402E +06 copies/. mu.L, and the Ct value of the 11 th group was 36.8 at a reduced concentration of 2.158E +02 copies/. mu.L. The results show that: the results of the primer probe sets of bacillus and staphylococcus are positive, and the results of the other primer probe sets are negative, which indicates that the primer probe composition provided by the invention has good specificity.

Example 4 sample testing

Randomly selecting 10 breast milk sample genome DNAs for testing, wherein the specific sample information is as follows: the genomic DNA concentration of S1 breast milk is 15.4 ng/. mu.L, the genomic DNA concentration of S2 breast milk is 2.2 ng/. mu.L, the genomic DNA concentration of S3 breast milk is 13.9 ng/. mu.L, the genomic DNA concentration of S4 breast milk is 102.4 ng/. mu.L, the genomic DNA concentration of S5 breast milk is 1.9 ng/. mu.L, the genomic DNA concentration of S6 breast milk is 172.3 ng/. mu.L, the genomic DNA concentration of S7 breast milk is 19.9 ng/. mu.L, the genomic DNA concentration of S8 breast milk is 69.3 ng/. mu.L, the genomic DNA concentration of S9 breast milk is 8.4 ng/. mu.L, and the genomic DNA concentration of S10 breast milk is 45.6 ng/. mu.L. The primer and probe sets, and the reaction system and reaction procedure were the same as in example 1, and the results of detection are shown in FIG. 6, and "/" in FIG. 6 indicates that no value was detected.

According to the detection result of the negative sample, the qualitative detection judgment rule of the sample to be detected is as follows: negative if Ct value is more than 36; less than 36 are positive. The results in FIG. 6 show that: the results of the probe 3, the probe 7, the probe 8 and the internal reference in the S1 sample are positive; the results of the probe 3, the probe 7, the probe 8, the probe 11 and the internal reference in the S2 sample are positive; the results of the probe 7, the probe 8, the probe 11 and the internal reference in the S3 sample are positive; the results of the probe 3, the probe 5, the probe 7, the probe 8 and the internal reference in the S4 sample are positive; the results of the probe 5, the probe 7, the probe 8 and the internal reference in the S5 sample are positive; the results of the probe 5, the probe 7, the probe 8 and the internal reference in the S6 sample are positive; the results of the probe 3, the probe 5, the probe 7, the probe 8 and the internal reference in the S7 sample are positive; the results of the probe 7, the probe 8 and the internal reference in the S8 sample are positive; the results of the probe 7, the probe 8 and the internal reference in the S9 sample are positive; the results of probe 7, probe 8 and internal controls were positive in the S10 sample.

Example 5 genus copy number quantification of bacteria in samples

5.1. Genus copy number quantification of bacteria in a sample

S4, S6 and S7 in example 4 were selected as samples to be tested, and negative control and positive control were set, respectively. The primer and probe set reaction system and reaction procedure were the same as in example 1. In the quality test of the detection experiment, the negative control has no amplification curve, and the internal references of the positive control have normal amplification curves, which indicates that the quality control of the experiment passes.

Quantification of samples S4, S6 and S7: internal quality control of the sample: the internal references of the 3 samples S4, S6 and S7 all have Ct values, which indicates that the samples are qualified for extraction and have amplification reaction, and the quality control of the samples passes; the quantitative results obtained for 3 samples from the standard curve are shown in FIG. 7, where "-" in FIG. 7 indicates no fluorescence signal was detected. In fig. 7, S4 indicates the breast milk samples to be tested, S4, S6 and S7 indicate the breast milk samples to be tested, NTC indicates a negative control, and PC indicates a positive control. Negative controls are not amplified, positive controls and reference genes are amplified, each sample can qualitatively detect whether 10 target species are contained or not at the same time, and the positive species can be quantitatively analyzed.

5.2. Second generation sequencing validation

5.2.1. Second Generation sequencing library construction

5.2.1.1.16SV4 region amplification

The S4, S6, and S7 genomes were used as templates and amplified using bacterial 16SV4 region primers Universal 519F (10. mu.M) and Universal 806R (10. mu.M).

The primer is synthesized in Shanghai, and the specific sequence is as follows:

Universal_519F：5'- cagcmgccgcggtaatwc-3'（SEQ ID No.35）；

Universal_806R：5'- ggactacvsgggtatctaat-3'（SEQ ID No.36）；

in the sequence, m is a or c, w is a or t, v is a or g or c, and s is g or c;

the amplification system is as follows: 2 x Phanta Max Master Mix (nuo kean, cat number P515-02), 15 μ L; universal _519F (10. mu.M), 1. mu.L; universal _806R (10. mu.M), 1. mu.L; about 50ng of genomic DNA; nuclease-free water was replenished to 30. mu.L.

The amplification procedure was:

the first stage is as follows: denaturation at 95 deg.C for 3min, 1 cycle;

and a second stage: denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, and 30 cycles;

and a third stage: extension at 72 ℃ for 5min for 1 cycle;

a fourth stage: storing at 4 ℃.

5.2.1.2. Magnetic bead purification

The PCR product harvested at 5.2.1.1 was purified using DNA sorting magnetic beads (Novozan, cat # N411-02). The purification steps are as follows:

1) after the magnetic beads are uniformly mixed, balancing for 30min at room temperature for later use;

2) adding 20 mu l of magnetic beads into the amplification product obtained in the previous step, and mixing the mixture by brief vortex;

3) performing instantaneous centrifugation, and standing at room temperature for 5min to fully combine the DNA with the magnetic beads;

4) placing the PCR tube on a magnetic frame, standing until the solution becomes clear (about 2 min), and removing the supernatant to avoid touching the magnetic beads;

5) adding 170 mu L of freshly prepared 80% absolute ethyl alcohol, incubating for 30s, and sucking and removing supernatant liquid to avoid touching magnetic beads;

6) repeating the step 5);

7) opening the cover to volatilize ethanol (from light reflection to frosted state) for about 3 min;

8) taking down from the magnetic frame, adding 42.5 μ l of nuclease-free water, mixing by vortex, centrifuging instantly, and standing at room temperature for 5 min;

9) the PCR tube was placed on a magnetic stand, left to stand until the solution became clear (about 2 min), and 40. mu.L of the supernatant was taken to a new PCR tube to avoid touching the magnetic beads.

5.2.2. Library enrichment (adding linker sequence)

5.2.2.1.PCR amplification

Taking 8 μ l of the purified product of the last step as a template for amplification, wherein the amplification system is as follows: 2 KAPA Hifi hotspot ready mix (KAPA, cat No. kk 2602), 25 μ L; i5 (nuo ke, cat N322), 1.5 μ L; i7 (nuo ke, cat N322), 1.5 μ L; product of 5.2.1, 8 μ L; the enzyme-free nucleic acid solution was filled to 50. mu.L.

The amplification procedure was:

the first stage is as follows: denaturation at 95 deg.C for 3min, 1 cycle;

and a second stage: denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, 6 cycles;

and a third stage: extension at 72 ℃ for 5min for 1 cycle;

a fourth stage: storing at 4 ℃.

5.2.2.2. purification of PCR products

The 5.2.2.1 harvested PCR product was purified using DNA sorting magnetic beads (Novozam, cat # N411-02). The purification steps are as follows:

2) adding 50 mu L of nuclease-free water into the PCR system to complement to 100 mu L;

3) adding 60 mul of magnetic beads into 100 mul of amplification products, and uniformly mixing by vortex;

4) performing instantaneous centrifugation, and standing at room temperature for 5min to fully combine the DNA with the magnetic beads;

5) placing the PCR tube on a magnetic frame, standing until the solution becomes clear (about 2 min), transferring the supernatant to a new PCR tube to avoid touching magnetic beads;

6) adding 20 mu L of magnetic beads into the supernatant, and uniformly mixing by vortex;

7) performing instantaneous centrifugation, and standing at room temperature for 5min to fully combine the DNA with the magnetic beads;

8) placing the PCR tube on a magnetic frame, standing until the solution becomes clear (about 2 min), and absorbing and removing the supernatant;

9) adding 170 mu L of freshly prepared 80% absolute ethyl alcohol, standing at room temperature for 30s, and removing supernatant to avoid touching magnetic beads;

10) repeating step 9);

11) the cover is opened to volatilize the ethanol (from light reflection to frosting) for about 3min, so that the magnetic beads are prevented from being dried excessively;

12) taking down from the magnetic frame, adding 42 μ L nuclease-free water, mixing by vortex, centrifuging instantly, and standing at room temperature for 5 min;

13) place the PCR tube on a magnetic stand, stand until the solution becomes clear (about 3 min), take 40. mu.L of supernatant to a new 1.5mL EP tube, avoid touching the magnetic beads.

Qubitlibrary quantitation

Mu.l of Invitrogen premix (Invitrogen, cat. No. Q33231) was added to 2. mu.L of the library, and the library concentration was determined at Qubit4.0.

5.2.2.4. Library sequencing

A sequencing instrument: illumina NovaSeq;

sequencing strategy: PE 150;

sequencing data quantity: each sample was not lower than 20000 Reads.

5.2.3. Second generation sequencing data analysis

5.2.3.1. Data analysis

The original Data (Raw Data) obtained by sequencing has a certain proportion of interference Data (Dirty Data), and in order to make the result of information analysis more accurate and reliable, the original Data is firstly spliced and filtered to obtain effective Data (clear Data).

The original sequencing data contains joint information, low-quality bases and undetected bases (expressed by N), the joint information, the low-quality bases and the undetected bases can cause great interference on subsequent information analysis, the interference information is removed through a fine filtering method, and finally obtained data are effective data and are called clean data or clean reads. The detection result is shown in fig. 8, wherein Effective Rate (%) = Clean Reads/Raw Reads; q20 refers to a base quality value of greater than 20 (i.e., a percentage of data with a sequencing error rate of less than 0.01 per base).

5.2.3.2. Relative abundance of species

The relative abundance of the species generated from the information alignment is shown in FIG. 9, for example, for the graduation ratios of the bacteria to be detected in the three breast milk samples. In fig. 9, the first column of the table represents the sample number, the first row represents the detected bacterial genus, and the numerical value represents the abundance of bacterial genus (i.e., percentage in all data).

The second generation sequencing results of the S4 sample showed that the abundance of Corynebacterium, stenotrophomonas, Bacillus, and Aquifex were 5.00%, 6.09%, 28.08%, and 4.52%; the second generation sequencing results of the S6 sample showed that the abundance of stenotrophomonas was 5.59%, the abundance of Bacillus was 38.54%, and the abundance of Megaster was 25.48%; the second generation sequencing results of the S7 sample showed that the abundance of Corynebacterium, stenotrophomonas, Bacillus and Aquifex were 6.70%, 27.39% and 5.45%, respectively. The analysis can be used for obtaining that the result of the sample determined by using the primer probe composition provided by the invention is basically consistent with the result of the second generation sequencing.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Edwardsies (Beijing) medical laboratory Co., Ltd

<120> kit for detecting 10 bacteria in human breast milk by fluorescence quantitative method and application thereof

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5862

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420

tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg 480

taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 540

atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 600

ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 660

taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 720

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 780

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 840

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 900

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 960

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1020

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1080

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1140

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1200

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1620

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1680

tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1740

acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1800

tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1860

ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1920

agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1980

tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2040

acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2100

agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2160

actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2220

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2280

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2340

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2400

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2460

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2520

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2580

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2640

gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2700

ccctttcgtc ttaaagcctg aagaattgac ggatttaacc gcacaagaaa ttctttaccg 2760

tttatacaat gaagaagaag ttcgtctgcc agacgttgaa cagcttcaat tcaattgcac 2820

ctgttcacgc gataaatgca aaaatgccga gaacaagact tgggcaaagg acgcgccgcc 2880

ttcgaacagt ggcaggacgc ggcgcacaac attttcagcg agcagctgcc ggccgatgac 2940

gacgccgctt tcgatttccg gctcaacttc acgcggttta tcgcagcaga gggtagctga 3000

ggctctggga gtctcgcggc agacggttat cagcatcgaa aaaggccgct acgatccttc 3060

cttgcctcta gcttttcatg attccttgca gtacgaccac ttcgaggcag gccagcgtta 3120

tcgcctggag gggcgctcgt tgtcctacac accgaatatc cggctgtaca acgccgcgcg 3180

tcagttattg gcgcaggagc gcagcctgct gccagccagg accattgcgc tgcaccgcat 3240

tcggcggcca tggccggatg ctgcgacgat gcagcatcaa cggtctgcca gagcaatgag 3300

tgcgaatgcc cgccggcaca ggaggattcc gactagacct cttacttgag ataactaaga 3360

tagctcgtgc aacttactat tatcaactaa agaaactgaa taaaccagat aaagacaaag 3420

caatcaaatc tgacattcaa tccagacttg agtgcagaag aggaaagtgg aattccatgt 3480

gtagcggtga aatgcgtaga gatatggagg aacaccagtg gcgaaggcga ctttctggtc 3540

tgtaactgac actgaggcac tgggcgtaaa gcgagtgtag gtggctcatt aagtcacatg 3600

tgaaatcccc gggcttaacc tgggaactgc atgtgatact ggtggtgcta gaatatgtga 3660

gagggaagta gaattccagg tgtaaaaagg ctttagcgtt ggtgttgtta gcattatctg 3720

cggctgccac agcagatacc tttgaatgcg atgcggtatc tcagcttaac caccctgccc 3780

ccaccgatac atgcgtggct ggaaagctaa cgaaaatatt gatttcaact tctttgatgc 3840

tcatgatttg aagccgctta cagatcgtgc aggagaagat acggttaaga ggcggctgag 3900

ggaaagtcgc tgaaccattg gccgttgttg acgcctgtct aacacctgta ctagctccaa 3960

ttaccactat tagaagtatg cttggcatca ttgatgcaaa gttgtacgag aagatagact 4020

ccatcctggc ctcgctgtcc accttccagc agatgtggat cagcaagcag gagtatgacg 4080

agtccggccc ctccatcgtc caccgcaaat gcttctaggc ggactatgac ttagttgcgt 4140

tacacccttt cttgacaaaa cctaacttgc gcagaaaaca agatgagatt ggcatggctt 4200

tatttgtttt ttttgttttg ttttggtttt tttttttttt ttggcttgac tcaggattta 4260

aaaactggaa cggtgaaggt gacagcttaa agcctgaaga attgacggat ttaaccgcac 4320

aagaaattct ttaccgttta tacaatgaag aagaagttcg tctgccagac gttgaacagc 4380

ttcaattcaa ttgcacctgt tcacgcgata aatgcaaaaa tgccgagaac aagacttggg 4440

caaaggacgc gccgccttcg aacagtggca ggacgcggcg cacaacattt tcagcgagca 4500

gctgccggcc gatgacgacg ccgctttcga tttccggctc aacttcacgc ggtttatcgc 4560

agcagagggt agctgaggct ctgggagtct cgcggcagac ggttatcagc atcgaaaaag 4620

gccgctacga tccttccttg cctctagctt ttcatgattc cttgcagtac gaccacttcg 4680

aggcaggcca gcgttatcgc ctggaggggc gctcgttgtc ctacacaccg aatatccggc 4740

tgtacaacgc cgcgcgtcag ttattggcgc aggagcgcag cctgctgcca gccaggacca 4800

ttgcgctgca ccgcattcgg cggccatggc cggatgctgc gacgatgcag catcaacggt 4860

ctgccagagc aatgagtgcg aatgcccgcc ggcacaggag gattccgact agacctctta 4920

cttgagataa ctaagatagc tcgtgcaact tactattatc aactaaagaa actgaataaa 4980

ccagataaag acaaagcaat caaatctgac attcaatcca gacttgagtg cagaagagga 5040

aagtggaatt ccatgtgtag cggtgaaatg cgtagagata tggaggaaca ccagtggcga 5100

aggcgacttt ctggtctgta actgacactg aggcactggg cgtaaagcga gtgtaggtgg 5160

ctcattaagt cacatgtgaa atccccgggc ttaacctggg aactgcatgt gatactggtg 5220

gtgctagaat atgtgagagg gaagtagaat tccaggtgta aaaaggcttt agcgttggtg 5280

ttgttagcat tatctgcggc tgccacagca gatacctttg aatgcgatgc ggtatctcag 5340

cttaaccacc ctgcccccac cgatacatgc gtggctggaa agctaacgaa aatattgatt 5400

tcaacttctt tgatgctcat gatttgaagc cgcttacaga tcgtgcagga gaagatacgg 5460

ttaagaggcg gctgagggaa agtcgctgaa ccattggccg ttgttgacgc ctgtctaaca 5520

cctgtactag ctccaattac cactattaga agtatgcttg gcatcattga tgcaaagttg 5580

tacgagaaga tagactccat cctggcctcg ctgtccacct tccagcagat gtggatcagc 5640

aagcaggagt atgacgagtc cggcccctcc atcgtccacc gcaaatgctt ctaggcggac 5700

tatgacttag ttgcgttaca ccctttcttg acaaaaccta acttgcgcag aaaacaagat 5760

gagattggca tggctttatt tgtttttttt gttttgtttt ggtttttttt ttttttttgg 5820

cttgactcag gatttaaaaa ctggaacggt gaaggtgaca gc 5862

<210> 2

<211> 24

<212> DNA

<213> Acinetobacter (Acinetobacter)

<400> 2

gcctgaagaa ttgacggatt taac 24

<210> 3

<211> 22

<212> DNA

<213> Acinetobacter (Acinetobacter)

<400> 3

tttgcattta tcgcgtgaac ag 22

<210> 4

<211> 24

<212> DNA

<213> Acinetobacter (Acinetobacter)

<400> 4

tctgccagac gttgaacagc ttca 24

<210> 5

<211> 20

<212> DNA

<213> Bifidobacterium (Bifidobacterium)

<400> 5

acaagacttg ggcaaaggac 20

<210> 6

<211> 19

<212> DNA

<213> Bifidobacterium (Bifidobacterium)

<400> 6

tgagccggaa atcgaaagc 19

<210> 7

<211> 20

<212> DNA

<213> Bifidobacterium (Bifidobacterium)

<400> 7

aaatgttgtg cgccgcgtcc 20

<210> 8

<211> 21

<212> DNA

<213> Corynebacterium (Corynebacterium)

<400> 8

gtttatcgca gcagagggta g 21

<210> 9

<211> 19

<212> DNA

<213> Corynebacterium (Corynebacterium)

<400> 9

agctagaggc aaggaagga 19

<210> 10

<211> 24

<212> DNA

<213> Corynebacterium (Corynebacterium)

<400> 10

cggcagacgg ttatcagcat cgaa 24

<210> 11

<211> 17

<212> DNA

<213> Stenotrophomonas (Stenotrophoromonas)

<400> 11

gccagccagg accattg 17

<210> 12

<211> 18

<212> DNA

<213> Stenotrophomonas (Stenotrophoromonas)

<400> 12

cgggcattcg cactcatt 18

<210> 13

<211> 23

<212> DNA

<213> Stenotrophomonas (Stenotrophoromonas)

<400> 13

cgacgatgca gcatcaacgg tct 23

<210> 14

<211> 22

<212> DNA

<213> Streptococcus (Streptococcus)

<400> 14

aggattccga ctagacctct ta 22

<210> 15

<211> 25

<212> DNA

<213> Streptococcus (Streptococcus)

<400> 15

gaatgtcaga tttgattgct ttgtc 25

<210> 16

<211> 30

<212> DNA

<213> Streptococcus (Streptococcus)

<400> 16

cttgagataa ctaagatagc tcgtgcaact 30

<210> 17

<211> 21

<212> DNA

<213> Bacillus (Bacillus)

<400> 17

tgagtgcaga agaggaaagt g 21

<210> 18

<211> 23

<212> DNA

<213> Bacillus (Bacillus)

<400> 18

agtgtcagtt acagaccaga aag 23

<210> 19

<211> 25

<212> DNA

<213> Bacillus (Bacillus)

<400> 19

tccatgtgta gcggtgaaat gcgta 25

<210> 20

<211> 19

<212> DNA

<213> genus Aquaculture (Enhydrosbacter)

<400> 20

gcgtaaagcg agtgtaggt 19

<210> 21

<211> 23

<212> DNA

<213> genus Aquaculture (Enhydrosbacter)

<400> 21

ctggaattct acttccctct cac 23

<210> 22

<211> 24

<212> DNA

<213> genus Aquaculture (Enhydrosbacter)

<400> 22

actgcatgtg atactggtgg tgct 24

<210> 23

<211> 22

<212> DNA

<213> Morganella (Morganella)

<400> 23

gctttagcgt tggtgttgtt ag 22

<210> 24

<211> 22

<212> DNA

<213> Morganella (Morganella)

<400> 24

cagggtggtt aagctgagat ac 22

<210> 25

<211> 25

<212> DNA

<213> Morganella (Morganella)

<400> 25

cgcattcaaa ggtatctgct gtggc 25

<210> 26

<211> 18

<212> DNA

<213> Providence genus (Providencia)

<400> 26

tacatgcgtg gctggaaa 18

<210> 27

<211> 18

<212> DNA

<213> Providence genus (Providencia)

<400> 27

cctcagccgc ctcttaac 18

<210> 28

<211> 24

<212> DNA

<213> Providence genus (Providencia)

<400> 28

ttgaagccgc ttacagatcg tgca 24

<210> 29

<211> 19

<212> DNA

<213> genus Staphylococcus (Staphylococcus)

<400> 29

gaaccattgg ccgttgttg 19

<210> 30

<211> 24

<212> DNA

<213> genus Staphylococcus (Staphylococcus)

<400> 30

cttctcgtac aactttgcat caat 24

<210> 31

<211> 26

<212> DNA

<213> genus Staphylococcus (Staphylococcus)

<400> 31

acgcctgtct aacacctgta ctagct 26

<210> 32

<211> 20

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 32

ctccatcctg gcctcgctgt 20

<210> 33

<211> 20

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 33

gctgtcacct tcaccgttcc 20

<210> 34

<211> 20

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 34

cctccatcgt ccaccgcaaa 20

<210> 35

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ccctacacga cgctcttccg atctcagcmg ccgcggtaat wc 42

<210> 36

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gttcagacgt gtgctcttcc gatctggact acvsgggtat ctaat 45

Claims

1. A multiplex PCR primer probe composition for identifying or assisting in identifying bacteria in breast milk is characterized in that: the bacteria are bacteria of 10 or less of the 10 genus bacteria of the genus Bifidobacterium, Acinetobacter, Corynebacterium, Streptococcus, Staphylococcus, Morganella, Provedorsia, Bacillus, stenotrophomonas, and Aquifex, and the multiplex PCR primer probe composition includes 1-FRP, 2-FRP, 3-FRP, 5-FRP, 6-FRP, 7-FRP, 8-FRP, 9-FRP, 10-FRP, and 11-FRP;

2. The multiplex PCR primer probe composition of claim 1 wherein: the primer 1-FP, the primer 1-RP, the Probe 1-Probe, the primer 2-FP, the primer 2-RP, the Probe 2-Probe, the primer 3-FP, the primer 3-RP, the Probe 3-Probe, the primer 5-FP, the primer 5-RP, the Probe 5-Probe, the primer 6-FP, the primer 6-RP, the Probe 6-Probe, the primer 7-FP, the primer 7-RP, the Probe 7-Probe, the primer 8-FP, the primer 8-RP, the Probe 8-Probe, the primer 9-FP, the primer 9-RP, the Probe 9-Probe, the primer 10-FP, the primer 10-RP, the Probe 2-FP, the primer 2-RP, the Probe 3-FP, the Probe 3-RP, the Probe 5-FP, the primer 5-RP, the Probe 5-Probe, the primer 6-FP, the Probe 6-RP, the Probe 7-FP, the Probe 7-RP, the Probe 7-Probe, the primer 8-FP, the Probe 8-RP, the Probe 8-Probe, the Probe 9-Probe, the Probe 10-FP, The amounts of the Probe 10-Probe, the primer 11-FP, the primer 11-RP and the Probe 11-Probe were the same.

3. The multiplex PCR primer probe composition according to claim 1 or 2 wherein: the multiplex PCR primer Probe composition also comprises an internal reference-FRP, wherein the internal reference-FRP is a specific primer and a Probe of an endogenous internal reference, and consists of a primer internal reference-FP, a primer internal reference-RP and a Probe internal reference-Probe, the primer internal reference-FP is single-stranded DNA shown in SEQ ID No.32, the primer internal reference-RP is single-stranded DNA shown in SEQ ID No.33, and the Probe internal reference-Probe is single-stranded DNA shown in SEQ ID No. 34.

4. The multiplex PCR primer probe composition according to claim 3 wherein: the amount of the substances of the primer internal reference-FP, the primer internal reference-RP and the Probe internal reference-Probe is the same as that of the substances of the primer 1-FP.

5. A reagent or a kit for identifying or assisting in identifying bacteria in breast milk, characterized in that: the reagent or kit contains the multiplex PCR primer probe composition of any one of claims 1 to 4.

6. The reagent or kit of claim 5, wherein: the reagent or the kit also comprises a positive standard substance, wherein the positive standard substance is a vector containing a DNA fragment with a nucleotide sequence shown as 4287-5862 site of SEQ ID No. 1.

7. Use of the multiplex PCR primer probe composition according to any one of claims 1 to 4 for the preparation of an identification or assisted identification of bacterial products in breast milk.

8. Use of the multiplex PCR primer probe composition of any one of claims 1 to 4 for identifying or aiding in identifying bacteria in breast milk; the use is for non-disease diagnostic and therapeutic purposes.

9. Use of the reagent or kit for identifying or aiding in the identification of bacteria in breast milk of claim 5 or 6 for identifying or aiding in the identification of bacteria in breast milk; the use is for non-disease diagnostic and therapeutic purposes.

10. A method for identifying bacteria in breast milk, comprising: the method comprises using genomic DNA of breast milk to be tested as a template, performing real-time fluorescence quantitative PCR detection by using the multiplex PCR primer probe composition of any one of claims 1 to 4 or the reagent or kit of claim 5 or 6, and determining which genus or genera of the 10 genus bacteria in claim 1 the breast milk to be tested contains; the methods are methods for non-disease diagnostic and therapeutic purposes.