CN115786550A - Primer combination, kit and method for detecting bacteria in low-microbial-mass biological sample - Google Patents

Abstract

The invention discloses a primer combination, a kit and a method for detecting bacteria in a low-microbial-mass biological sample, and belongs to the technical field of biology. The primer combination consists of primers with nucleotide sequence shown in SEQ ID No. 1-SEQ ID No. 10. The invention further discloses a kit and a method for detecting bacteria in a biological sample with low microbial biomass. By utilizing the primer combination, the kit and the method, the sensitivity and the detectable rate of detecting the bacterial flora in the low-microbial-mass biological sample can be effectively improved, the tolerance of the degraded sample is higher, and the qualified rate of detecting the bacterial flora in the degraded sample can be effectively improved.

Description

Primer combination, kit and method for detecting bacteria in low-microbial-mass biological sample

Technical Field

The invention relates to the technical field of biology, in particular to a primer combination, a kit and a method for detecting bacteria in a low-microbial-biomass biological sample.

Background

Existing studies have well documented that the human microbiome group is closely related to human health maintenance and disease development. High-throughput sequencing on high-variable regions such as V3 and V4 regions of a 16S rRNA gene sequence is a common and mature means for detecting high-abundance bacterial flora at present, and is widely applied to researches on intestinal flora, oral flora, vaginal flora, skin flora and the like. However, the effect of applying the 16S rRNA gene (V3V 4) to the detection of bacterial flora in a sample such as a tumor tissue is not satisfactory.

Detection of bacterial populations in tumor tissues meets these challenges: the bacterial biomass in the tumor tissue is low and the proportion of DNA belonging to the bacteria is very low in the extraction of DNA from the tissue. These challenges can result in bacteria within the tumor tissue being difficult to detect. The existing sample for detecting low bacterial biomass by 16S rRNA gene sequencing (V3-V4) has limited sensitivity and is not suitable for detecting the bacterial population in the tumor tissue.

Disclosure of Invention

In order to solve one of the above technical problems, the present invention aims to provide a highly sensitive primer and method for detecting bacteria in tumor tissue, which adopts multiple specific primers and multiple PCR methods, and combines with second-generation high-throughput sequencing to identify bacteria in tumor tissue. In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a primer combination for detecting bacteria in a low-microbial-quantity biological sample, which comprises a first primer pair consisting of a first upstream primer with a nucleotide sequence shown by SEQ ID No.1 and a first downstream primer with a nucleotide sequence shown by SEQ ID No.2, a second primer pair consisting of a second upstream primer with a nucleotide sequence shown by SEQ ID No.3 and a second downstream primer with a nucleotide sequence shown by SEQ ID No.4, a third primer pair consisting of a third upstream primer with a nucleotide sequence shown by SEQ ID No.5 and a third downstream primer with a nucleotide sequence shown by SEQ ID No.6, a fourth primer pair consisting of a fourth upstream primer with a nucleotide sequence shown by SEQ ID No.7 and a fourth downstream primer with a nucleotide sequence shown by SEQ ID No.8, and a fifth primer pair consisting of a fifth upstream primer with a nucleotide sequence shown by SEQ ID No.9 and a fifth downstream primer with a nucleotide sequence shown by SEQ ID No. 10.

In the present invention, the first, second, third, fourth and fifth primer pairs target V2, V3, V5, V6 and V8 regions, respectively, of a sequence of a bacterial 16S rRNA gene.

In some embodiments of the invention, each of the forward and reverse primers further has a sequencing universal primer sequence to create a library for identifying bacterial composition in a low microbial biomass biological sample using high throughput sequencing.

In a second aspect, the present invention provides a kit for detecting bacteria in a low microbial biomass biological sample, comprising a primer combination according to the first aspect of the present invention.

Further, the kit also comprises a DNA extraction reagent. In some embodiments of the invention, the DNA extraction reagent comprises a cleavage reagent including, but not limited to CTAB, lysozyme, and protease.

Still further, the kit further comprises PCR amplification reagents and/or PCR purification reagents. In some embodiments of the invention, the PCR amplification reagents comprise a DNA polymerase. In some preferred embodiments of the invention, the DNA polymerase, mg ²⁺ And dNTPs are premixed together.

In a third aspect, the invention provides the use of the primer combination of the first aspect of the invention in the preparation of a kit for detecting bacteria in a low microbial biomass biological sample based on high throughput sequencing.

In a fourth aspect, the present invention provides a method for detecting bacteria in a low microbial biomass biological sample, comprising the steps of:

s1, obtaining a DNA sample of the low microbial biomass biological sample;

s2, carrying out PCR amplification on the DNA sample by using the primer combination of claim 1;

s3, detecting whether a target band is amplified or not by using agarose gel electrophoresis, if so, recovering and purifying the target band to perform high-throughput sequencing,

and S4, comparing the high-throughput sequencing data with a bacterial 16S rRNA gene database, and identifying the bacterial species existing in the low-microbial-mass biological sample.

In some embodiments of the invention, the primer combination is in a PCR amplification system, and the final concentration of each primer is 0.2. Mu.M.

In some embodiments of the invention, the procedure of PCR amplification is: 2min at 95 ℃; 30sec at 94 ℃, 1min at 70 ℃, 40sec at 72 ℃ and 35 cycles; 5min at 72 ℃; keeping at 4 ℃.

In the present invention, the low microbial biomass biological sample is at least one selected from the group consisting of blood, urine, tissue, and cells.

In some embodiments of the invention, the tissue may be freshly obtained tissue, or may be a Formalin-fixed paraffin-embedded (FFPE) sample of tissue. In some embodiments of the invention, the tissue may be normal tissue or tumor tissue.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

by utilizing the primer combination, the kit and the method, the sensitivity and the detection rate of detecting the bacterial flora in the low-microbial-mass biological sample can be effectively improved.

By utilizing the primer combination, the kit and the method, the tolerance on the degraded sample is higher, and the qualified rate of flora detection in the degraded sample can be effectively improved.

Drawings

FIG. 1 shows the results of the amplification of the V2, V3, V5, V6, V8 single variable region primer pairs. M:100bp DNA Ladder;1: amplifying the result of the V2 region primer pair; 2: amplifying the result of the primer pair in the V3 region; 3: the amplification result of the V5 region primer pair; 4: amplifying the result of the primer pair in the V6 region; 5: and (3) amplifying the result by using the V8 region primer pair.

FIG. 2 shows the results of amplification of 2 variable region primer pairs of V2, V3, V5, V6, V8. M:100bp DNA Ladder;1: V2V3 amplification results; 2: V2V5 amplification results; 3: V2V6 amplification results; 4: V2V8 amplification results; 5: V3V5 amplification results; 6: V3V6 amplification results; 7: V3V8 amplification results; 8: V5V6 amplification results; 9: V5V8 amplification results; 10: V6V8 amplification results.

FIG. 3 shows the results of amplification of 3 variable region primer pairs from V2, V3, V5, V6, V8. M:100bp DNA Ladder;1: (iv) V2V3V5 amplification results; 2: (iv) V2V3V6 amplification results; 3: (iv) V2V3V8 amplification results; 4: V3V5V6 amplification results; 5: (iv) V3V5V8 amplification results; 6: (iv) V5V6V8 amplification results; 7: (iv) V2V5V6 amplification results; 8: (iv) V2V5V8 amplification results; 9: (iv) V2V6V8 amplification results; 10: V3V6V8 amplification results.

FIG. 4 shows the results of amplification of 4 variable region primer pairs of V2, V3, V5, V6, V8. M:100bp DNAladder;1: the amplification result of the V1/V2/V3/V4 area primer pair; 2: the amplification result of the V1/V2/V3/V4 area primer pair; 3: the amplification result of the V1/V2/V3/V4 area primer pair; 4: the amplification result of the primer pair of the V1/V2/V3/V4 region; 5: and (3) amplifying the result by using the primer pair of the V1/V2/V3/V4 region.

FIG. 5 shows the results of V2, V3, V5, V6, V8 variable region primer pair amplification. M:100bp DNA Ladder;1: and (3) amplifying the primer pair of the V1/V2/V3/V4/V5 region.

FIG. 6 shows the results of the amplification of the V3/V4 region primer pair. M:100bp DNAladder;1: the amplification result of the primer pair of the V1/V2/V3/V4/V5 area in the sample 1; 2: the amplification result of the primer pair for V1/V2/V3/V4/V5 region in sample 2.

FIG. 7 shows the results of library amplification with V2, V3, V5, V6, V8 variable region primer pairs. M:100bp DNA Ladder;1: amplifying a library result by using a V1/V2/V3/V4/V5 region primer pair in the sample 1; 2: amplifying a library result by using a V1/V2/V3/V4/V5 region primer pair in the sample 2; 3: negative control (NTC).

FIG. 8 shows that sequencing of the amplified pool of 5 variable region primers identified bacteria at the genus level within the tumor tissue.

Detailed Description

Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety, and the equivalent family of patents is also incorporated by reference, especially where relevant in the art to which these documents disclose definitions of relevant terms. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For ranges containing a numerical value less than 1 or containing a fraction greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is considered to be 0.0001,0.001,0.01, or 0.1, as appropriate. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be expressed and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not intended to exclude the presence of other elements, steps or procedures not expressly disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Rather, the term "consisting essentially of 8230 \8230; \8230composition" excludes any other components, steps or processes from the scope of any of the terms hereinafter recited, insofar as they are necessary for performance. The term "consisting of 823070 \8230composition" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clear, the present invention is further described in detail below with reference to the embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The molecular biology experiments, which are not specifically described in the following examples, were carried out according to the specific methods listed in the manual "molecular cloning laboratory Manual" (fourth edition) (J. Sambulu., M.R. Green, 2017), or according to the kit and product instructions. Other experimental methods, unless otherwise specified, are conventional. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

EXAMPLE 1 design of specific primers

The inventors specifically designed 5 pairs of specific primers according to the sequence characteristics of the 16S rRNA gene of bacteria, as shown in Table 1:

TABLE 15 pairs of specific primers for the rapid identification of bacteria in tumor tissues

F1 and R1 amplify V2 for 16S, F2 and R2 amplify V3, F3 and R3 amplify V5, F4 and R4 amplify V6, and F5 and R5 amplify V8.

By using the multiplex PCR amplification technology, the above 5 pairs of primers can simultaneously amplify and construct an amplification library of 16S 5 variable regions (V2, V3, V5, V6 and V8), and then perform second-generation sequencing to identify the bacterial composition in the tumor tissue (the primers are designed with universal primers required for sequencing).

5 primers at the F end need to be premixed, the primers are diluted to 5 mu M, 20 mu L of each primer is mixed together, each primer is 1 mu M after mixing, 5 primers at the R end need to be premixed, the primers are diluted to 5 mu M, 20 mu L of each primer is mixed together, and each primer is 1 mu M after mixing.

The primer can be prepared into a kit for detecting bacteria in tumor tissues.

Example 2 validation of primers

To test the effectiveness of the 5 pairs of primers to amplify the 16S rRNA gene, fecal DNA was used as a sample and the example primer pairs were used to amplify single variable regions, 2 variable regions, 3 variable regions, 4 random combinations of variable regions, and 5 variable regions of V2, V3, V5, V6, V8 of the 16S rRNA gene, respectively. The 16S rRNA gene was amplified as a positive control with a universal V3V4 primer pair (341F-CCTACGGGNGGCWGAG, 805R-GACTACHVGGGTATCTAATCC) and the target fragment was approximately 600bp (FIG. 6). The amplification results of the single variable region, 2 variable regions, 3 variable regions, 4 variable regions, and 5 variable regions are shown in FIGS. 1 to 5, respectively, and the amplification results of the positive control are shown in FIG. 6. The results show that the primer pairs of 5 variable regions can successfully amplify the target band of the 16S rRNA gene regardless of single amplification or combined amplification, and the more variable regions are amplified, the brighter the target band is obtained by amplification.

Example 3 detection of bacteria in tumor tissue

1. Preparation of multiplex PCR reaction System:

(1) Reaction solution: 2 × Phanta Max master mix;

(2) Primer solution: the forward and reverse primers were mixed to give 1. Mu.M each single primer.

The bacteria in the tumor tissue were identified by the reaction system described above in the following manner.

2. Tumor tissue DNA extraction:

1) Adding a proper amount of beads into a centrifuge tube, and taking a proper amount of sample (50-100 mg).

2) To the centrifuge tube containing the sample was added 1mL of 65 ℃ preheated CTAB extract, 50. Mu.L lysozyme, 20. Mu.L protease, the setting of the grinder was 50Hz, grinding was carried out for 4min, and a 65 ℃ water bath was carried out for 60min.

3) Shaking up for 2-3 times during the water bath period to ensure full cracking. After being taken out, the mixture is cooled to room temperature.

4) Pipette the solution from the tube into a new 2.0mL sterile centrifuge tube.

5) 800. Mu.L of chloroform-isoamyl alcohol (24.

6) Aspirate 600 μ L of supernatant into a new 2mL sterile centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (24).

7) Pipette 400. Mu.L of supernatant into a new 1.5mL sterile centrifuge tube, add 2/3 of the volume of the supernatant isopropanol and 1/10 of the volume of sodium acetate (3M), mix well, and stand at-20 ℃ for 1h.

8) Centrifuging at 12000rpm for 10min, discarding supernatant, centrifuging instantaneously, sucking off excessive liquid with a gun head, and air drying at room temperature for 10min until DNA precipitate is semitransparent.

9) Adding 50-100 μ L of sterile ddH containing 10mg/mL RNase ₂ And O, dissolving the DNA precipitate, digesting in a water bath kettle at 37 ℃ for 1 hour, taking supernatant, transferring the supernatant into another centrifugal tube to be used as template DNA, and storing the rest sample liquid in a refrigerator at-20 ℃.

3. Multiplex PCR amplification reaction:

(1) The multiplex PCR amplification system is shown in Table 2.

TABLE 2 multiplex PCR amplification System

Components	Volume (μ L)
		Sample DNA to be amplified	7.5
Primer mixture	5
		2×Phanta Max master mix	12.5
Total volume	25

1) Taking out 2X Phanta master mix, thawing and centrifuging;

2) Calculating the required volume, mixing the required volume and the mixed primers in an eight-connection pipe, performing vortex centrifugation, and placing in a refrigerator at 4 ℃ for later use;

3) Preparing a new 96-well plate, and adding the enzyme and primer mixture into the 96-well plate;

4) Adding corresponding sample DNA to be amplified, and sealing plates;

5) The PCR reaction was performed with the procedure: 2min at 95 ℃; 30sec at 94 ℃, 1min at 70 ℃, 40sec at 72 ℃ and 35 cycles; 5min at 72 ℃; keeping at 4 ℃.

And 4, PCR product purification:

1) Adding 40 mu L10 mM Tris-HCl into 25 mu L PCR product, adding 50 mu L magnetic beads, blowing, beating and mixing for 15 times, and standing for 5min at room temperature;

2) Placing the centrifuge tube in a magnetic rack for 5min until the liquid is clear, and discarding the supernatant;

3) Keeping the centrifuge tube on a magnetic frame, adding 200 μ L of freshly prepared 80% ethanol, and paying attention to not disturb the magnetic beads; standing for 30sec, and sucking and discarding the supernatant;

4) Adding 200 μ L of fresh 80% ethanol again, and discarding the supernatant after 30 sec;

5) Leaving the centrifuge tube on a magnetic frame, and airing for 3-5min at room temperature;

6) Adding 50 μ L10 mM Tris-HCl, blowing and mixing for 15 times, standing at room temperature for 5min;

7) The centrifuge tube was placed on a magnetic rack for 5min until the liquid was clear. The supernatant (library) was transferred to a new 96-well plate and the library was stored at-20 ℃.

5. And (3) judging an amplification result:

the multiplex PCR products were run on agarose gel electrophoresis, and as shown in FIG. 7, the major fragments were concentrated to about 450 to 480bp (containing the sequencing universal primer sequence). The bacterial species present in the tumor tissue can be identified by combining NovaSeq 6000PE250 sequencing and comparison with the 16S rRNA gene database, and the results are shown in FIG. 8.

As can be seen from FIG. 8, the combination of five pairs of primers can identify the bacterial flora in tumor tissue.

Example 4 detection of amplification yield

To further verify the sensitivity and detection rate of the primers designed in the examples of the present invention, the inventors amplified 10 tumor tissue samples with single variable region primer pairs of V2, V3, V5, V6, V8, 2 variable regions, 3 variable regions, 4 variable region primer pairs randomly combined, and 5 variable region primer pairs combined, and at the same time, amplified the V3V4 primer pair as experimental control.

The PCR product was detected by agarose gel electrophoresis using the target band amplified as a standard result of amplification eligibility, and the results are shown in Table 3.

TABLE 3 percent of pass of amplification of bacterial flora in tumor tissue by primers in 5 regions

As can be seen from Table 3, the combination of five pairs of V2, V3, V5, V6 and V8 primers for bacterial detection of tumor tissue samples has higher sensitivity and amplification yield.

EXAMPLE 5 detection of degraded tumor tissue samples

The inventors used 10 paraffin-embedded tumor tissue samples (FFPE) of DNA (with varying degrees of degradation), and amplified bacterial sequences in degraded samples using V2, V3, V5, V6, V8 variable regions in combination with V3V4 primer pairs, respectively, to amplify the target band as a criterion for eligibility.

The amplification results are shown in Table 4.

TABLE 4 qualification rate of bacterial flora in FFPE sample amplified by combining 5 variable region primer pairs

Primers for amplification	Amplification yield (%)
		V2, V3, V5, V6 and V8 primer pairs are combined together	70％
V3V4 primer pair	30％

As can be seen from the data in the table, the combination of five pairs of primers V2, V3, V5, V6 and V8 for bacterial detection of FFPE samples has higher DNA degradation tolerance and amplification yield.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A primer combination for detecting bacteria in a low-microbial-quantity biological sample is characterized by comprising a first primer pair consisting of a first upstream primer with a nucleotide sequence shown by SEQ ID No.1 and a first downstream primer with a nucleotide sequence shown by SEQ ID No.2, a second primer pair consisting of a second upstream primer with a nucleotide sequence shown by SEQ ID No.3 and a second downstream primer with a nucleotide sequence shown by SEQ ID No.4, a third primer pair consisting of a third upstream primer with a nucleotide sequence shown by SEQ ID No.5 and a third downstream primer with a nucleotide sequence shown by SEQ ID No.6, a fourth primer pair consisting of a fourth upstream primer with a nucleotide sequence shown by SEQ ID No.7 and a fourth downstream primer with a nucleotide sequence shown by SEQ ID No.8, and a fifth primer pair consisting of a fifth upstream primer with a nucleotide sequence shown by SEQ ID No.9 and a fifth downstream primer with a nucleotide sequence shown by SEQ ID No. 10.

2. The primer combination of claim 1, wherein each of the forward primer and the reverse primer further comprises a sequencing universal primer sequence.

3. A kit for detecting bacteria in a low microbial biomass biological sample, comprising the primer combination of claim 1 or 2.

4. The kit of claim 3, further comprising a DNA extraction reagent.

5. The kit of claim 3, further comprising PCR amplification reagents and/or PCR purification reagents.

6. Use of the primer combination of claim 1 for the preparation of a kit for the detection of bacteria in a low microbial biomass biological sample based on high throughput sequencing.

7. A method for detecting bacteria in a low microbial biomass biological sample, comprising the steps of:

s1, obtaining a DNA sample of the low microbial biomass biological sample;

s2, performing PCR amplification on the DNA sample by using the primer combination of claim 1;

s3, detecting whether a target band is amplified or not by using agarose gel electrophoresis, if so, recovering and purifying the target band to perform high-throughput sequencing,

and S4, comparing the high-throughput sequencing data with a bacterial 16S rRNA gene database, and identifying the bacterial species existing in the low-microbial-mass biological sample.

8. The method of claim 7, wherein the primer combination is used in a PCR amplification system at a final concentration of 0.2. Mu.M for each primer.

9. The method of claim 7, wherein the PCR amplification procedure is: 2min at 95 ℃; 30sec at 94 ℃, 1min at 70 ℃, 40sec at 72 ℃ and 35 cycles; 5min at 72 ℃; keeping at 4 ℃.

10. The method of any one of claims 7 to 9, wherein the low microbial biomass biological sample is at least one selected from the group consisting of blood, urine, tissue, and cells.

Images