阅读说明：本技术 一种高通量低成本的微量生物样品分子鉴定技术 (High-throughput low-cost molecular identification technology for trace biological samples ) 是由 彭华正 金群英 朱汤军 叶华琳 于 2020-04-28 设计创作，主要内容包括：本发明涉及一种分子生物领域,尤其涉及一种高通量低成本的微量生物样品分子鉴定技术,包括高质量基因组提取和序列检测分析这两方面内容。本发明旨在降低微量生物样品分子鉴定的高通量测序成本。上述的一种高通量低成本的微量生物样品分子鉴定技术,步骤如下：1)样品收集；2)样品裂解；3)核酸收集；4)带标记引物的PCR扩增；5)PCR产物混合测序和位置还原。本发明不需要制备复杂的原生质体,受体来源简单,适合没有孢子的真菌转化。另外,本发明简化和提升了现有的DNA提取技术,使研究者能够利用实验室的一些简单小型设备,方便地开展大规模的高质量基因组提取；同时整合普通PCR和高通量测序技术,结合计算机分析,对上述提取的DNA实现低成本的PCR序列扩增和测序分析。(The invention relates to the field of molecular biology, in particular to a high-throughput low-cost molecular identification technology for a trace biological sample, which comprises two aspects of high-quality genome extraction and sequence detection analysis. The invention aims to reduce the high-throughput sequencing cost of molecular identification of trace biological samples. The molecular identification technology for the trace biological sample with high flux and low cost comprises the following steps: 1) collecting a sample; 2) cracking a sample; 3) collecting nucleic acid; 4) PCR amplification of the marked primer; 5) PCR products were mixed for sequencing and site reduction. The invention does not need to prepare complicated protoplast, has simple receptor source and is suitable for the transformation of the fungus without spores. In addition, the invention simplifies and improves the existing DNA extraction technology, so that researchers can conveniently carry out large-scale high-quality genome extraction by using some simple and small-sized devices in a laboratory; meanwhile, common PCR and high-throughput sequencing technologies are integrated, and the PCR sequence amplification and sequencing analysis with low cost are realized on the extracted DNA by combining computer analysis.)

1. a high-throughput low-cost molecular identification technology for trace biological samples is characterized by comprising the following steps:

(1) collecting samples: collecting a micro-microorganism sample in a small centrifugal tube, and removing a liquid part of the sample containing liquid through short-time high-speed centrifugation;

(2) sample lysis: adding glass beads with the diameter of 0.1mm-0.5mm to 1/8 of the total volume of the tube, adding protease K suspension with the concentration of 1mg/ml of the total volume of the tube 1/8, preserving the temperature at 40-70 ℃ for 5-30 minutes, and immediately performing horizontal or vertical oscillation at the speed of more than 1000 rpm;

(3) nucleic acid collection: adding 5-10% chelex solution (1/4 tubes) in total volume, keeping at 80-95 deg.C for 10-30min, performing high speed freezing and centrifuging for a short time, and freezing and storing supernatant;

(4) PCR amplification with labeled primers: amplifying each extracted DNA sample with primers with different combinations of labels;

(5) PCR product mix sequencing and site reduction: and mixing PCR products of all tubes, performing high-throughput sequencing, and reducing all sequences into sample tubes at all positions through different primer marking combinations of all tubes.

2. The high throughput low cost micro biological sample molecular characterization technique of claim 1, wherein: the term "micro-biological sample" as used herein refers to a wide variety of biological samples that are readily manipulated by a micropipette, including a wide variety of animal and plant cell masses, fungi, and bacteria, including gram-negative and positive bacteria.

3. The high throughput low cost micro biological sample molecular characterization technique of claim 1, wherein: the extraction vessels can be operated simultaneously in multiple single tube operations, 12-tube, 96-well plate, or 384-well plate devices, enabling high throughput and low cost.

4. The high throughput low cost micro biological sample molecular characterization technique according to claim 1, wherein step (4) comprises the following steps:

designing universal upstream and downstream primers according to a conserved region of a gene sequence to be amplified;

adjusting 1-2 bases at the non-5' end of the primer to form different primer combinations corresponding to different PCR tube positions;

adding a joint of 30-35 bases at the 5' end of the primer, and connecting the joint with PCR amplification in subsequent high-throughput sequencing, and simultaneously further enhancing the amplification stability;

each sample is amplified separately by primers with different labeled sites.

5. The high-throughput low-cost micro biological sample molecular identification technology according to claim 1, characterized in that the specific content of the step (5) is as follows:

after amplification, 1-5 mul of products are respectively taken from each tube to be mixed, and the mixed products are used as a sample to be subjected to high-throughput sequencing;

hundreds of thousands of sequences obtained by sequencing are identified by computer software at the labeled sites, and the sequences are redistributed to each sample tube.

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a high-throughput low-cost molecular identification technology for a trace biological sample, which comprises two aspects of high-quality genome extraction and sequence detection and analysis of the trace biological sample.

Background

Modern molecular biotechnology is gradually entering the era of high-throughput processing in the aspects of DNA sample extraction, PCR amplification and sequencing, which is very necessary in many research fields, for example, when the diversity of certain biological specific gene loci is researched by molecular means, a large number of samples (including animal and plant cells and microorganisms) must be usually faced, and the high-throughput processing usually depends on expensive automatic processing machines and kits. For laboratory studies under ordinary conditions, if genome extraction, PCR amplification and sequencing are performed manually one by using a kit according to a conventional method, time and labor are wasted, and the cost is often difficult to bear. In terms of DNA extraction, it is not difficult to extract high quality DNA of a single sample using a kit, and improvement and optimization of the method are required for performing rapid low-cost extraction of samples in batches. In recent years, many researchers have proposed improvements in DNA extraction methods, but often only one kind of microorganism sample, such as only gram-negative bacteria, is subjected to difficulty in extracting various samples simultaneously, such as intestinal bacteria including gram-negative bacteria which are relatively easy to extract DNA, and gram-positive bacteria such as bifidobacterium which are difficult to extract DNA; moreover, most of the previous improvements tend to oversimplify the extraction process, seriously reducing the quality of the extracted DNA, in order to meet the needs of large scale extraction. In terms of sequence detection analysis, although PCR and classical sequencing methods are mature and reliable, it is time-consuming and labor-consuming to sequence thousands of samples, so that high-throughput sequencing is staggering, but the sequencing cost of each sample is not cheap and the use cost is high.

Disclosure of Invention

In order to solve the technical problems so as to economically and rapidly carry out molecular identification of trace biological samples based on high flux, the invention provides two innovative contents: the first is to simplify and improve the existing DNA extraction technology, so that researchers can conveniently, economically and rapidly extract high-quality genomes in a large scale by using some simple and small devices in a laboratory; secondly, integrating common PCR and high-throughput sequencing technology, and combining computer analysis to realize low-cost PCR sequence amplification and sequencing analysis on the extracted DNA;

the invention adopts the following technical scheme to realize the first innovation:

preferably, the technical scheme comprises the following steps:

(1) the samples are treated according to the dry and wet states: directly picking a relatively dry sample such as a microbial colony and the like into a centrifugal tube to carry out the next step; if the suspension is liquid cell suspension, centrifuging at 4 deg.C 10000g for 1min, removing supernatant, washing with sterile water, centrifuging at 4 deg.C 10000g for 1min, and removing supernatant; the pipe can be selected from calandria and 96-hole plate;

(2) adding glass beads with diameter of 0.1mm-0.5mm to 1/8 of the total volume of the tube according to the volume of the tube, adding protease K suspension with concentration of 1mg/ml of the total volume of 1/8 tubes, preserving the temperature at 40-70 ℃ for 5-30 minutes, and horizontally or vertically shaking at the speed of more than 1000rpm for 5 min;

(3) adding 10% chelex solution with total volume of 1/4 tubes, scattering, cooling at 95 deg.C for 10-20min, and ice-cooling;

(4) centrifuging at 4 deg.C 10000g for 1min, taking supernatant as template, and freezing for storage;

the invention adopts the following technical scheme to realize the second innovation content:

preferably, the technical scheme comprises the following steps:

(1) designing primer marker sites corresponding to different PCR tubes;

(2) PCR amplification and sample mixed sequencing;

(3) reducing the positions of the PCR tubes where different primers are positioned according to the detected primer sequences;

preferably, the step (1) includes the following three steps:

designing universal upstream and downstream primers according to the conserved region of the gene sequence to be amplified, wherein the length is 20-25 bases, the Tm value is 55-60 ℃, and the length of an amplification product is 300-500 bp;

② introducing 1-2 mismatched bases as specific markers between the 5' end of the upstream and downstream primers and the middle region of the primers, wherein the upstream primer designs 24 mismatched combinations, and the downstream primer designs 16 mismatched combinations, thus forming 384 different primer combinations which can correspond to 384 different PCR tube positions;

③ adding 30-35 base joint at the 5' end of the primer, linking the PCR amplification in the subsequent high-flux sequencing, so that the length of the primer is increased to about 45 base, and the amplification stability can be further enhanced;

preferably, the step (2) includes the following three steps:

setting a PCR reaction system to be 15 mu l, and respectively mixing PCR reaction reagents required by each row and each line in proportion, wherein the PCR reaction reagents comprise primers;

combining rows and columns, preparing a reaction system of each hole, wherein the reaction system comprises different primer combinations, and finally adding templates into each hole respectively;

③ PCR reaction, mixing 1-5 μ l of products in each well, marking as a sample, and carrying out Illumina Miseq deep sequencing;

preferably, the step (3) includes the following two steps:

① identifying the relative mark of pipe hole position and classifying by computer software, wherein the identification requires that 1-2 adjusting bases at upstream and downstream positions can be selected when appearing, to eliminate error to the maximum;

② sequencing the sequence measured in each well, so that the reliability of more sequence readings is higher, the sequence with less readings can be further verified by a classical sequencing method, the sequence number is less than the set prediction, and the sequence with less readings is taken as an error to be eliminated, for example, the sequence in each well can be reduced to the maximum extent;

the invention is divided into a microorganism genome extraction part and a PCR sequence amplification and sequence analysis part. The extracted partial characteristics of the microbial genome are as follows: the method which is suitable for high-throughput operation, such as glass bead oscillation, enzymolysis of protease K, chelating resin, high temperature and the like, is utilized to cooperatively play a role, so that a large number of microbial samples can be released with high-quality DNA in a short time; the PCR sequence amplification and sequence analysis technology is characterized in that different PCR samples are marked by using a primer combination, then the one-time sequencing of mixing different samples is realized by using a deep sequencing technology, and finally the primer marks are sorted by using a computer technology; the two parts of technologies can be tightly connected, so that the high-flux and low-cost operation in the whole process is realized, and each part can also be used as one part of other molecular biology identification technologies;

compared with the existing genome extraction technology and PCR sequence analysis technology, the invention has the following beneficial effects:

(1) only a small amount of samples are needed to extract trace DNA, for example, colonies are picked from a flat plate, so that the trouble of preparing a large amount of strains is avoided;

(2) the whole operation can be carried out by a plurality of single tubes, and the operation is more suitable for the operation of high-throughput equipment such as a microporous plate and the like;

(3) the method is particularly suitable for large-scale strain identification and diversity research of specific gene loci in the flora;

(4) the extracted genome has higher quality, and the stability of PCR sequence amplification is ensured;

the characteristics of high flux and low cost are reflected by the genome extraction technology, the PCR sequence amplification and the sequence analysis.

Drawings

FIG. 1 gram-positive bacterium Bifidobacterium longum(Bifidobacterium.longum) The extraction effect in 8-PCR tubes;

FIG. 2 PCR amplification effect of enteric bacteria.

Detailed Description