Bacterium 16S amplification kit, detection method and application

Abstract

The invention relates to the technical field of bacterium 16S detection, in particular to a bacterium 16S amplification kit, a detection method and application. The kit comprises a universal and universal bacterium 16S amplification reagent, and further comprises a PCR (Polymerase Chain Reaction) reaction solution and an additive. The reagent can be used for high-throughput 16S amplification, the amplification success rate can be improved for different samples and different sources, sample differences do not need to be considered, and automatic sample loading and high-throughput sequencing are achieved.

Description

Technical Field

[0001] The present application relates to the technical field of bacterial 16S detection, and in particular to a bacterial 16S amplification kit, a detection method and an application. Background Art

[0002] Traditional bacterial culture and biochemical analysis can no longer meet the diagnosis and epidemiological research of various pathogenic microorganisms. Researchers not only hope to study various bacteria and viruses at the molecular level, but also hope that the test methods will be faster, more accurate, and have higher throughput. Human factors should be reduced as much as possible to facilitate the construction of standardized operating procedures. Since 16SrDNA sequences exist in all bacterial species and can meet the requirements for comparison, 16SrDNA sequence analysis is used as the basis for virulence and toxin gene identification methods to identify unknown bacteria in the shortest time and make accurate judgments on emerging bacterial infectious diseases.

[0003] Currently, the existing bacterial 16S amplification reagents are limited by success rate, specificity, yield, cost and throughput, and cannot be adapted and adjusted to different bacteria, which inevitably increases the detection cost. Summary of the invention

[0004] To this end, the present application embodiment provides a bacterial 16S amplification kit with stronger adaptability and higher amplification success rate. The kit adds additives to the reaction system, which can cause poor amplification specificity for the 16S sample part with a large amount of host genome, improve product specificity, and improve amplification success rate. To this end, the present application embodiment discloses the following technical solutions:

[0005] On the one hand, the present application embodiment discloses a bacterial 16S amplification kit, comprising:

[0006] Universal primer pair for amplifying bacterial 16S rDNA;

[0007] A PCR reaction solution, wherein the PCR reaction solution comprises a PCR buffer, 2 mM MgCl2, 0.2 mM dNTPs and 2 U / μL Taq DNA polymerase; and

[0008] The additive is selected from at least one of 0.4-1.0 μg / μL betaine, 0.4-1.0 μg / μL bovine serum albumin and 0.2-2.0 mM tetramethylammonium chloride.

[0009] In the examples of the present application, the nucleotide sequences of the universal primer pairs are shown as SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 and SEQ ID NO.4.

[0010] In the examples of the present application, the additive is one of 0.4-1.0 μg / μL betaine, 0.4-1.0 μg / μL bovine serum albumin or 0.2-2.0 mM tetramethylammonium chloride.

[0011] In the embodiment of the present application, the additive is betaine and 0.4-1.0 μg / μL bovine serum albumin; or

[0012] The additive is 0.4-1.0 μg / μL bovine serum albumin and 0.2-2.0 mM tetramethylammonium chloride; or

[0013] The additives are betaine and 0.2-2.0 mM tetramethylammonium chloride.

[0014] In the examples of the present application, the additives are betaine, 0.4-1.0 μg / μL bovine serum albumin and 0.2-2.0 mM tetramethylammonium chloride.

[0015] On the one hand, the present application embodiment discloses a method for detecting bacterial 16S, comprising:

[0016] extracting bacterial DNA from the bacterial suspension using an extraction reagent;

[0017] The bacterial DNA is added as a template to a PCR reaction system comprising universal primers, a PCR reaction solution and additives to amplify the V3V4 variable region of the bacterial 16S rDNA by PCR; and

[0018] The amplified products were subjected to agarose electrophoresis, and the bacterial 16S band was determined based on the electrophoresis;

[0019] Wherein, the nucleotide sequence of the universal primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 and SEQ ID NO.4.

[0020] The PCR reaction solution comprises PCR buffer, 2mM MgCl2, 0.2mM dNTPs and 2U / μL TaqDNA polymerase; the additive is selected from at least one of 0.4-1.0μg / μL betaine, 0.4-1.0μg / μL bovine serum albumin and 0.2-2.0mM tetramethylammonium chloride.

[0021] In an embodiment of the present application, the PCR amplification system comprises 25 μL of: 1 μL of template, 1 μL of each of the upstream and downstream primers of the universal primer, and 12.5 μL of a mixed solution consisting of the PCR reaction solution and the additive, and the remainder is double distilled water; the PCR amplification conditions are: 98°C 45s; 98°C 10s, 55°C 60s, 72°C 30s, 72°C 5min, 12°C∞; wherein, 98°C 10s, 55°C 60s and 72°C 30s constitute a cycle, for a total of 30 cycles.

[0022] On the one hand, the embodiments of the present application also disclose the application of the 16S amplification kit in the detection of bacterial 16S in water sludge, leaf filter membranes, air filter membranes, water filter membranes, jejunum, rectum, cecum, fish intestinal contents, pearl grouper hindgut tissue, livestock feces, mouse feces, human feces, human intestinal contents and soil.

[0023] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0024] The kit provided in the embodiment uses additives to perform 16S amplification on different samples containing bacteria, and can accurately amplify the target fragment. The kit can be used to effectively detect bacteria in water sludge, leaf filter membranes, air filter membranes, water filter membranes, jejunum, rectum, cecum, fish intestinal contents, pearl grouper hindgut tissue, livestock feces, mouse feces, human feces, human intestinal contents and soil, and has the advantages of low cost, easy operation and high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Agarose gel electrophoresis diagram of the PCR amplification product of bacterial 16S detected by the kit provided in the embodiment of the present application in leaf filter membrane, air filter membrane and water filter membrane.

[0026] Figure 2 Agarose gel electrophoresis diagram of the PCR amplification product of bacterial 16S detected by the kit provided in the examples of the present application in the jejunum, rectum, cecum, fish intestinal contents, pearl grouper hindgut tissue, and human intestinal contents.

[0027] Figure 3 Agarose gel electrophoresis diagram of PCR amplification products of bacterial 16S in livestock feces, mouse feces, and human feces detected by the kit provided in the examples of the present application.

[0028] Figure 4 This is an agarose gel electrophoresis diagram of the PCR amplification product of bacterial 16S in water sludge detected by the kit provided in the embodiment of the present application.

[0029] Figure 5This is an agarose gel electrophoresis diagram of the PCR amplification product of bacterial 16S in soil detected by the kit provided in the examples of the present application. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0031] The embodiment of the present application provides a bacterial 16S amplification kit with stronger adaptability and higher amplification success rate. The kit includes: a universal primer pair for amplifying bacterial 16S rDNA; a PCR reaction solution, the PCR reaction solution includes a PCR buffer, 2mM MgCl2, 0.2mM dNTPs and 2U / μL TaqDNA polymerase; and an additive, the additive is selected from at least one of 0.4-1.0μg / μL betaine, 0.4-1.0μg / μL bovine serum albumin and 0.2-2.0mM tetramethylammonium chloride.

[0032] The kit can be used to identify the target bands amplified by 16S primers for samples from different scenarios, such as leaf filters, air filters, water filters, jejunum, rectum, cecum, fish intestinal contents, pearl grouper hindgut tissue, livestock feces, mouse feces, human feces, human intestinal contents and soil, to determine the excellent amplification capability of the kit.

[0033] Preparation of the kit

[0034] (1) Universal primer pair, such as SEQ ID NO: 1-2, 10 μM, synthesized by Shanghai Bioengineering Co., Ltd.;

[0035] (2) PCR reaction solution:

[0036] PCR buffer contains: 0.1% (v / v) NP-40, 0.02% (v / v) gelatin (purchased from Sigma, USA), 0.1% (v / v) Tween-20 (purchased from Sigma, USA), 0.06M pH=8.9 Tricine (purchased from Merck), 100mM KCl (purchased from Sigma), 10mM NH4Cl (purchased from Sigma)

[0037] MgCl2: purchased from Sigma, USA.

[0038] 0.2 mM dNTPs: purchased from Shanghai Dingguo Biotechnology Co., Ltd.

[0039] 2U / μL DNA polymerase: purchased from ABclonal.

[0040] (3) Additives

[0041] Betaine and bovine serum albumin were purchased from Sigma, USA, and prepared at 0.4, 0.6, 0.8, and 1.0 μg / μL, respectively;

[0042] Tetramethylammonium chloride was purchased from Sigma, USA, and prepared into 0.2, 0.5, 1.0, 1.5 and 2.0 mM respectively.

[0043] Testing of different samples

[0044] 1. Instrument

[0045] Bio-Rad S1000PCR instrument, Beckman Microfuge 22R desktop micro-refrigerated centrifuge, Beijing Liuyi agarose gel electrophoresis instrument, Shanghai Peiqing gel imaging system, QIAGEN PyroMark Q96ID sequencer.

[0046] 2. Sample pretreatment

[0047] Leaf filters, air filters, water filters, jejunum, rectum, cecum, fish intestinal contents, pearl grouper hindgut tissue, human intestinal contents, livestock feces, mouse feces, human feces, water sludge and soil samples were collected respectively.

[0048] Add an equal amount of liquefaction reagent to the sample, shake to mix, and let stand at room temperature for 10 to 20 minutes; pipette 1 mL of liquefied sample solution into a 1.5 mL sterile centrifuge tube, centrifuge at 13,000 rpm for 10 min, and remove the supernatant; add 1 mL of washing reagent to the precipitate, shake to mix, centrifuge at 13,000 rpm for 10 min, and remove the supernatant; repeat once and take the supernatant, which is the bacterial suspension.

[0049] 3. Extraction of bacterial DNA

[0050] The obtained DNA was used as a template to perform PCR amplification of bacterial 16S rDNA using universal primers;

[0051] The PCR amplification system contains 25 μL of the following: 1 μL of template, 1 μL of upstream and downstream primers of the universal primer, and 12.5 μL of a mixture consisting of the PCR reaction solution and the additive, and the remainder is double distilled water. The PCR amplification conditions are: 98°C 45s; 98°C 10s, 55°C 60s, 72°C 30s, 72°C 5min, 12°C∞; wherein 98°C 10s, 55°C 60s and 72°C 30s constitute a cycle, for a total of 30 cycles.

[0052] The DNA sequence of the corresponding fragment obtained by PCR was imported into the standard nucleic acid sequence database for sequence comparison and identification of the bacterial strain.

[0053] Add the cut nucleic acid amplification product to the sol solution in the gel recovery kit, dissolve it completely in a water bath, add magnetic beads to absorb the nucleic acid fragments, wash it twice with 80% alcohol, dry it, and collect the purified nucleic acid. The purified nucleic acid is sequenced and aligned. It should be noted that the method for preparing agarose gel is to weigh 1.5g of agarose and place it in 100mL TAE electrophoresis buffer, heat it to melt, and when the temperature drops to about 60°C, evenly spread it on a plate to make a 1% agarose gel.

[0054] Under the same conditions, the test results are as follows: Figures 1 to 5 As shown, specific target bands can be effectively amplified, which indicates that the amplification efficiency of the reagents of the present invention reaches 100%, which is much higher than the results of conventional multiple experiments;

[0055] In addition, using cost analysis, the above 13 samples, using traditional reagents, need to take 2 days; and the success rate needs to constantly adjust the input amount to achieve an 80% success rate for amplification PCR identification; the success rate of amplification PCR identification using the kit provided in this application exceeds 90%, and there is no need to adjust the input amount of each sample. It can be seen that this application has established a 16S nucleic acid detection method for common bacterial samples, and clarified the range and length of DNA characteristic sequences in 16S rDNA that have identification and classification significance. This application can be used to achieve rapid and integrated identification of bacterial contaminants, which solves the problem that the existing conventional technology requires a long time and has a high false positive test result, and provides strong technical support for the rapid detection of common bacterial microorganisms.

[0056] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A bacterial 16S amplification kit, characterized in that: include: Universal primer pair for amplifying bacterial 16S rDNA; A PCR reaction solution, wherein the PCR reaction solution comprises a PCR buffer, 2 mM MgCl2, 0.2 mM dNTPs and 2 U / μL TaqDNA polymerase; as well as The additive is selected from at least one of 0.4-1.0 μg / μL betaine, 0.4-1.0 μg / μL bovine serum albumin and 0.2-2.0 mM tetramethylammonium chloride.

2. The 16S amplification kit according to claim 1, characterized in that The nucleotide sequences of the universal primer pair are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The 16S amplification kit according to claim 1, characterized in that The additive is betaine, 0.4-1.0 μg / μL bovine serum albumin or 0.2-2.0 mM tetramethylammonium chloride.

4. The 16S amplification kit according to claim 1, characterized in that The additive is 0.4-1.0 μg / μL betaine and 0.4-1.0 μg / μL bovine serum albumin; or the additive is 0.4-1.0 μg / μL bovine serum albumin and 0.2-2.0 mM tetramethylammonium chloride; or the additive is 0.4-1.0 μg / μL betaine and 0.2-2.0 mM tetramethylammonium chloride.

5. The 16S amplification kit according to claim 1, characterized in that The additives are 0.4-1.0 μg / μL betaine, 0.4-1.0 μg / μL bovine serum albumin and 0.2-2.0 mM tetramethylammonium chloride.

6. A method for detecting bacterial 16S, characterized in that: include: extracting bacterial DNA from the bacterial suspension using an extraction reagent; The bacterial DNA is added as a template to a PCR reaction system comprising universal primers, PCR reaction solution and additives, and PCR amplification of the V3V4 variable region of the bacterial 16S rDNA is performed; and the amplified product is subjected to agarose electrophoresis. The bacterial 16S band is determined according to the electrophoresis; the nucleotide sequence of the universal primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 and SEQ ID NO.

4. The PCR reaction solution comprises PCR buffer, 2mM MgCl2, 0.2mM dNTPs and 2U / μL Taq DNA polymerase; the additive is selected from at least one of 0.4-1.0μg / μL betaine, 0.4-1.0μg / μL bovine serum albumin and 0.2-2.0mM tetramethylammonium chloride.

7. The detection method according to claim 6, characterized in that: The PCR amplification system, calculated as 25 μL, comprises: 1 μL of template, 1 μL of upstream and downstream primers of the universal primer, and 12.5 μL of a mixture consisting of the PCR reaction solution and the additive, and the remainder is double distilled water; The PCR amplification conditions are: 98°C 45s; 98°C 10s, 55°C 60s, 72°C 30s, 72°C 5min, 12°C∞; wherein 98°C 10s, 55°C 60s and 72°C 30s constitute a cycle, for a total of 30 cycles.

8. Use of the 16S amplification kit according to any one of claims 1 to 5 in the detection of bacterial 16S in leaf filters, air filters, water filters, jejunum, rectum, cecum, fish intestinal contents, pearl grouper hindgut tissue, human intestinal contents, livestock feces, mouse feces, human feces, water sludge, and soil.

Images