Multiple PCR primer pair combination for bacterium determination and determination method

A technology of primer pairs and bacteria, which is applied in biochemical equipment and methods, recombinant DNA technology, measurement/inspection of microorganisms, etc., can solve the problems of inability to distinguish bacteria, and achieve the effect of efficient identification

Inactive Publication Date: 2016-12-21
上海华点云生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

But Baker et al. [6] Point out that the current study cannot distinguish all bacteria by a single variable region

Method used

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  • Multiple PCR primer pair combination for bacterium determination and determination method
  • Multiple PCR primer pair combination for bacterium determination and determination method
  • Multiple PCR primer pair combination for bacterium determination and determination method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment one: the design of primer pair combination

[0053] 1. Design of primer pairs

[0054] According to the conserved region sequence of 16S rDNA, primer pairs were designed to amplify 8 variable regions, among which, the V1 and V2 regions were amplified using the P1 primer pair, the V3 and V4 regions were amplified using the P3 primer pair, and the V5 and V6 regions were amplified using the P5 primers For amplification, the V7 and V8 regions were amplified using the P6 primer pair (Table 1a, 1b).

[0055] In Table 1a and 1b, the underlined primer sequence is the sequence that binds to the 16S rDNA conserved region, and the primer sequence that is not underlined is the sequence that is complementary to the sequencing general primer of the Illumina Miseq V1-V2 kit. Between the sequence complementary to the sequencing primer and the sequence combined with the 16S rDNA conserved region, different tag sequences can be added according to the final library sample numb...

Embodiment 2

[0066] Example 2: Screening of primer pair combinations

[0067] 1. Extract bacterial nucleic acid

[0068] 1) Use the DNA Purification from Blood or Body Fluids (Spin Protocol) (Qiagen Cat.NO.51306) kit to extract the genomic DNA of Legionella pneumophila (purchased from Beijing Beinan Chuanglian Biotechnology Research Institute).

[0069] 2) Quantify the DNA in the collection tube with a spectrophotometer. The requirements for DNA used for flux detection are: concentration: ≥10ng / μl; purity: OD260 / 280 of 1.8-2.0; total amount of nucleic acid: ≥1μg. Short-term storage at -20°C; long-term storage at -80°C.

[0070] 2. PCR amplification

[0071] 1) Synthesize each primer in the P1, P3, P5, and P6 primer pairs and dilute them into 5 μM use solution;

[0072] In this example, there is only one sample, so select one tag sequence, which is the index sequence "TGAACCTT" provided by the Illumina sequencing platform; that is, the sequences of each primer in the P1, P3, P5, and P6 ...

Embodiment 3

[0100] Embodiment three: Utilize primer pair combination to identify bacteria

[0101] 1. Strains

[0102] Select 6 strains (Clostridium difficile, Enterococcus faecium, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Staphylococcus aureus, purchased from Beijing Beina Chuanglian Biotechnology Research Institute) as simulated experimental strains, used The determined optimal primer combination and the most suitable primer concentration were used for two-step PCR, and the strains were identified by high-throughput sequencing analysis.

[0103] 2. The first step of PCR amplification

[0104] The primer pair combinations P1 (5 μM)+P5 (5 μM); P3 (5 μM)+P6 (5 μM) were selected, and the sequences of each primer were the same as step 2 in Example 2. Experimental procedure Refer to step 2 in Example 2 to carry out the first step of PCR amplification.

[0105] 3. The first step of PCR product purification

[0106] a. configure 80% ethanol solution;

[0107]b. Add...

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Abstract

The invention belongs to the field of biological technologies and provides a multiple PCR primer pair combination for bacterium determination which can be used for next generation sequencing of bacteria and a bacterium determination method. The primer pair combination is utilized to amplify V1-V8variable zones of a bacterium 16S rDNA, effectively obtains variable zone sequences; the bacterial species can be sensitively and efficiently determined by combining next-generation sequencing and bioinformatic analysis.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for identifying bacteria. Background technique [0002] 16S rRNA is a subunit of ribosomal RNA, and 16S rDNA is the gene encoding this subunit. Due to the conservation and ubiquity of the 16S rDNA sequence, as well as the stability of the nucleic acid sequence itself, the reproducibility of sequence analysis is extremely high [1] . According to these conserved regions, universal primers for bacteria can be designed to amplify the 16S rDNA fragments of all bacteria, and these primers are only specific to bacteria, which means that these primers will not complement non-bacterial DNA. The difference in the 16S rDNA variable region of bacteria can be used to distinguish different bacteria. Therefore, 16S rDNA is considered the gold standard for bacterial identification. [0003] Bacterial 16S rDNA contains nine variable regions [2] , named as V1 ~ V9 area. Except for V2, V3, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689C12Q2600/16
Inventor 陈晨王海印
Owner 上海华点云生物科技有限公司
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