A pair of primers for Brucella 23S rRNA gene and its application

Abstract

The invention discloses a pair of primers for Brucella 23S rRNA gene and application thereof, and relates to the field of biological detection technology. The primers for the Brucella 23S rRNA gene are composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2. According to the characteristics of high abundance of Brucella 23S rRNA, the present invention designs primers in its conservative region, constructs a RT-qPCR detection method, and compares the detection limit with the qPCR method. The results show that compared with qPCR, the RT-qPCR detection method can reduce the Ct value by 2-3 values, significantly improving the sensitivity of the detection. The detection method of the present invention improves the sensitivity of Brucella detection and can detect Brucella with lower copies.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a pair of primers of Brucella 23S rRNA gene and application thereof. Background Art

[0002] Brucellosis is a chronic bacterial infectious disease caused by Brucella infection that affects humans and a variety of animals. It has a significant impact on the development of animal husbandry worldwide. The disease is characterized by rapid transmission and high infectiousness, which not only causes economic losses to the breeding industry, but also seriously threatens veterinary public health safety.

[0003] Bacteriology, serology, and molecular testing are three common methods for diagnosing brucellosis infection. Bacteriology-based diagnosis is a standard method, but it requires a culture cycle of at least 4-5 days for testing, strict protective facilities, and skilled technicians. The low specificity of Brucella immunogenic epitopes and their high similarity with Escherichia coli, Salmonella (SP), Yersinia (SP), Vibrio (SP), and other bacteria also limit the serological diagnosis of brucellosis. Molecular detection methods such as polymerase chain reaction (PCR) and real-time PCR (RT-PCR) have overcome the shortcomings of serology and bacterial culture diagnostic methods to some extent and have been widely used.

[0004] Existing diagnostic methods have evolved from conventional PCR to real-time quantitative PCR, which has greatly improved the sensitivity of diagnosis. However, after Brucella infection, some bacteria often lurk in macrophages and can reproduce and cause disease when the body's resistance is reduced. Therefore, improving the sensitivity of Brucella detection is a necessary measure to prevent and control brucellosis.

[0005] Ribosomes are ubiquitous in cells and their number is huge. Each prokaryotic cell generally contains about 15-18×10 3 ribosomes; each eukaryotic cell generally has 10 6 Up to 10 7 Cells with active protein synthesis (such as immature toad egg cells) often have more ribosomes than normal cells, with a content of up to about 10 per cell. 12 indivual.

[0006] Ribosomes are a type of ribonucleoprotein particle in cells, mainly composed of RNA (rRNA) and proteins. Their function is to convert genetic codes into amino acid sequences and construct protein polymers from amino acid monomers according to the instructions of mRNA. The bacterial ribosome 70S ribosome consists of a 30S small subunit and a 50S large subunit. The 30S small subunit contains 16S RNA (1540 nucleotides) and 21 ribosomal proteins; the large subunit consists of 5S RNA (120 nucleotides), 23S RNA (2900 nucleotides) and 31 ribosomal proteins. Bacterial 16S rRNA is highly conservative and is often used for bacterial classification. The 23S rRNA variability between different species is slightly stronger, and it is relatively rare to design primers based on it for bacterial identification. In order to improve the sensitivity of Brucella detection, the present invention intends to use 23s rRNA as the target gene to establish an RT-qPCR detection method for Brucella. Summary of the invention

[0007] The purpose of the present invention is to provide a pair of primers for Brucella 23S rRNA gene and application thereof, so as to solve the problems existing in the above-mentioned prior art. By using the primers for Brucella 23S rRNA gene provided by the present invention to perform RT-qPCR detection on Brucella, the sensitivity of Brucella detection can be improved, and Brucella with lower copies can be detected.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The invention provides a pair of primers for Brucella 23S rRNA gene, which are composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0010] The present invention also provides the use of the above-mentioned Brucella 23S rRNA gene primers in preparing products for RT-qPCR detection of Brucella.

[0011] Furthermore, the product is a kit or a reagent.

[0012] The present invention also provides a product for detecting Brucella by RT-qPCR, comprising the above-mentioned primers of Brucella 23S rRNA gene.

[0013] Furthermore, the product is a kit or a reagent.

[0014] The present invention also provides an application of the above-mentioned Brucella 23S rRNA gene primer or product in RT-qPCR detection of Brucella for non-disease diagnosis purposes.

[0015] The present invention also provides a RT-qPCR detection method for Brucella for non-disease diagnosis purposes, comprising the following steps:

[0016] Extract the total nucleic acid of the sample to be tested, and reverse transcribe to obtain cDNA;

[0017] The cDNA is used as a template and the primers of the Brucella 23S rRNA gene are used to perform qPCR amplification to obtain the Ct value of the sample to be tested. The Ct value of the sample to be tested is substituted into the standard curve equation to calculate the content of Brucella in the sample to be tested.

[0018] Furthermore, the reaction system of the qPCR amplification includes: 2×One Step SYBR Green Mix 10 μL, One Step SYBR Green Enzyme Mix 1 μL, 50×ROX Reference Dye II 0.4 μL, 10 μM upstream and downstream primers 0.4 μL each, template 1 μL and RNase-free water 6.5 μL.

[0019] Furthermore, the reaction conditions of the qPCR amplification include: 95°C for 30s; cycle reaction at 95°C for 10s, 52-54°C for 30s, for a total of 35 cycles; melting curve reaction at 95°C for 15s, 60°C for 60s, and 95°C for 15s.

[0020] Preferably, the reaction conditions of the qPCR amplification include: 95°C for 30s; cycle reaction at 95°C for 10s, 53°C for 30s, for a total of 35 cycles; melting curve reaction at 95°C for 15s, 60°C for 60s, and 95°C for 15s.

[0021] The present invention discloses the following technical effects:

[0022] The present invention establishes a detection method for Brucella, which is RT-qPCR with high-abundance 23S rRNA as template, and the method improves the sensitivity of pathogen detection, and the detection based on 23S rRNA can distinguish the growth state of bacteria, which can be used to determine the development trend of bacterial infection and the antibacterial effect of drugs. According to the characteristics of high abundance of Brucella 23SrRNA, the present invention designs primers in its conservative region, constructs a RT-qPCR detection method, and compares the detection limit with the qPCR method. The results show that compared with qPCR, the RT-qPCR detection method can reduce the Ct value by 2-3 numerical values, significantly improving the sensitivity of detection. In a word, the detection method of the present invention improves the sensitivity of Brucella detection, and can detect Brucella with lower copies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Amplification curves at different temperatures;

[0025] Figure 2 is the Ct value at different temperatures;

[0026] Figure 3 is the amplification curve of different template dilutions;

[0027] Figure 4 A standard curve is drawn based on dilutions of templates with different concentrations;

[0028] Figure 5 This is the gel electrophoresis diagram of the amplified product of the specific experiment;

[0029] Figure 6 Sequencing results of PCR products for specific experiments;

[0030] Figure 7 It is the statistical difference between qPCR and RT-qPCR samples before and after reverse transcription;

[0031] Figure 8 are the amplification curves of qPCR and RT-qPCR; 1 is RT-qPCR and 2 is qPCR;

[0032] Fig. 9 The statistical difference comparison results between the detection method established by the present invention and the standard IS711 detection method;

[0033] Fig.10 Real-time fluorescence quantitative PCR amplification curves of logarithmic phase samples before and after reverse transcription;

[0034] Fig.11 Real-time fluorescence quantitative PCR for stationary samples before and after reverse transcription;

[0035] Fig.12 It is the comparison result of Ct value before and after reverse transcription in stationary phase and before and after reverse transcription in logarithmic phase;

[0036] Fig.13 RT-qPCR test results of clinical samples;

[0037] Fig.14 These are the results of qPCR testing of clinical samples. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] Preparation of S1 sample template

[0045] S1.1 Take 200 μL of Brucella s2 vaccine strain, put it into a 1.5 mL centrifuge tube, and use the MiniBEST total nucleic acid extraction kit to extract total nucleic acid.

[0046] Establishment of Real-time Fluorescence Quantitative RT-PCR Detection Method for S2

[0047] S2.1 According to the 23S rRNA reference sequence (NR_103168.2) of Brucella abortus bv.1 strain 9-941 registered in GenBank, Brucella (taxid: 234) was defined in the reference RNA database and BLAST comparison analysis was performed. Then, primers were designed using the online program Prime-BLAST according to the conserved sequence, and a pair of amplification primers were designed (see Table 1), which amplified a nucleic acid fragment with a length of 182 bp.

[0048] Table 1 Primer sequences

[0049]

[0050] S2.2 Optimization of reaction temperature in detection method

[0051] The total reaction system of real-time fluorescence quantitative PCR was set to 20 μL. 2×One Step SYBR Green Mix 10 μL, One Step SYBR Green Enzyme Mix 1 μL, 50×ROX Reference Dye II 0.4 μL, upstream and downstream primers (10 μM) 0.4 μL each, template 1 μL, RNase-free water 6.5 μL were added to the RNase-free centrifuge tube in sequence to prepare a mixed solution for use. The real-time fluorescence PCR reaction conditions were: 95°C 30s; cycle reaction 95°C 10s, 52-54°C 30s, a total of 35 cycles; melting curve reaction 95°C 15s, 60°C 60s, 95°C 15s.

[0052] The amplification curves at different temperatures and the Ct values ​​at each temperature are shown in Figure 1 and Figure 2 ,Depend on Figure 2 From the Ct value results, it can be seen that the Ct value is the smallest in the range of 52-54℃, so 53℃ is selected as the measurement temperature of Brucella qPCR.

[0053] S2.3 Sensitivity analysis of the detection method

[0054] A nucleic acid sample of 14.932 ng / μL was diluted tenfold in sequence, for a total of seven orders of magnitude (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7) as qPCR amplification, the optimized method of the present invention was used for amplification. When the optimized reaction conditions were used to establish the standard curve, in the process of formulating the standard curve, according to the calculation copy formula: (6.02×10 23 = (MW:DNA bp×660) × (copy number / mol) × (concentration) / MW g / mol. Directly dilute the nucleic acid sample of known mass concentration with enzyme-free water. Draw the standard curve based on the results. The amplification curves of different template dilutions and the differences in the drawn standards are shown in Figure 3 and Figure 4 .

[0055] like Figure 3 As shown in Figure 2, the exponential phase amplification curves of different concentration gradient templates showed a good parallel relationship. In addition, the sample was diluted and the standard curve was drawn according to the Ct value of each concentration. The results are shown in Figure 2. Figure 4 , the horizontal axis represents the sample nucleic acid concentration, and the vertical axis represents the number of qPCR reaction cycles. The starting concentration of the nucleic acid sample is 14.932ng / μL, and the final copy number is 7.4e+10. After 7-fold dilution, the Ct value is 38.859, and the copy number is 7.4e+3, and no specific amplification is performed. Correlation coefficient R 2 The standard curve has a good linear relationship. The results show that the standard curve established by the present invention has high reliability and accuracy.

[0056] S2.4 Specificity analysis of detection methods

[0057] To verify the specificity of amplification, the specificity of primers was first analyzed in the Prime-BLAST bacterial database. Then, real-time quantitative PCR was performed using common clinical bacterial nucleic acids as templates. Melting curve analysis and electrophoresis analysis were performed after amplification. The amplified bands were sequenced and the sequences were analyzed by BLAST to determine the specificity of amplification.

[0058] The fluorescent PCR products were verified by agarose gel electrophoresis, and only the Brucella S2 vaccine strain showed a clear band. The amplified band was between 100 bp and 200 bp ( Figure 5 ). The target fragment was recovered by gel extraction, and the purified PCR product was sequenced. After amplification, the amplified product showed a single peak at a Tm value of about 84°C ( Figure 6 ), no nonspecific amplification peak.

[0059] S2.5 Determine whether reverse transcription can improve the sensitivity of Brucella detection

[0060] In order to determine whether reverse transcription can improve the sensitivity of Brucella detection, fluorescent quantitative PCR amplification with and without reverse transcription steps was performed using the total nucleic acid of Brucella as a template. The reaction system was the same as S2.2, and the reaction temperature was 53°C. The reverse transcription step was 55°C for 15 minutes. The sensitivity of the detection was determined by the size of the Ct value.

[0061] The total nucleic acid of Brucella was used as template to compare the fluorescence quantitative PCR amplification with and without the reverse transcription step. The results are shown in Figure 7 and Figure 8 . The results showed that after adding the reverse transcription step, the Ct value of the amplification reaction decreased by 2-3, indicating that the sensitivity of detection can be improved by reverse transcription when the total nucleic acid of Brucella is used as a template. The DNA content in the sample was compared with the cDNA content formed after reverse transcription of the total nucleic acid in the sample. Three samples were selected for the experiment, and the results showed that the Ct values ​​were significantly different. The Ct value of the reverse transcription step is 1-3 values ​​higher than the Ct value without the reverse transcription step. This shows that the cDNA content in the nucleic acid after reverse transcription is significantly higher than the DNA content in the total nucleic acid, so there is amplification of RNA in the template, which indirectly proves that its detection method is effective.

[0062] S2.6 Comparison between the detection method of the present invention and the general method

[0063] After nucleic acid extraction, two groups of experiments were performed, the first group was primers designed based on 23Sr RNA, and the second group was Brucella universal primer IS711. Real-time fluorescence quantitative PCR was performed using the above experimental conditions, and the Ct values ​​of the two groups of experiments were compared.

[0064] Comparing the Ct values ​​displayed by different primers, the detection method of the present invention can detect RNA in the sample and is more sensitive than IS 711. The 23S rRNA primer can reflect the difference in Ct values ​​before and after reverse transcription, while the universal primer does not reflect this difference ( Fig. 9 ).

[0065] S2.7 Comparison of bacterial logarithmic phase and stationary phase

[0066] Verify the RNA content of bacteria at different growth stages, and then explore the infection time of bacteria in vivo. Incubate the bacteria in a 37°C shaker overnight until the bacterial solution reaches OD 450 At 1.0, the RNA content of bacteria is high, and nucleic acid is extracted at this time. The extracted nucleic acid samples are quickly frozen in liquid nitrogen to ensure that the RNA in them is not degraded, and then the quick-frozen nucleic acid samples are stored at -80°C. The bacteria are stored at 4°C to allow the bacteria to enter a stationary period, and then nucleic acid is extracted from the bacteria.

[0067] Two nucleic acid samples were tested and compared:

[0068] There are obvious differences in the nucleic acid samples of bacteria in the logarithmic phase before and after reverse transcription. The average Ct value of the nucleic acid samples after logarithmic phase reverse transcription is 2 times higher than that of the nucleic acid samples without reverse transcription, which indirectly proves that there are more bacterial RNA in the logarithmic phase. In the stationary phase samples, there is little difference before and after reverse transcription, and the Ct value does not increase significantly ( Figure 10-12 ).

[0069] S2.8 Testing of naturally infected clinical specimens

[0070] The RT-qPCR and qPCR methods established in the present invention were used to detect the total nucleic acid of 16 blood clinical samples to observe the specificity and sensitivity of the newly established detection method. The results are shown in Fig.13 and Fig.14 .

[0071] The test results showed that two samples were negative and 14 samples were positive. There were 4 groups of samples whose Ct values ​​changed significantly before and after reverse transcription, with the Ct values ​​changing by 1-3 times.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A pair of RT-qPCR primers for detecting Brucella 23S rRNA gene, characterized in that: It consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

2.

2. Use of the RT-qPCR primer for detecting Brucella 23S rRNA gene as claimed in claim 1 in preparing a product for RT-qPCR detection of Brucella.

3. The use according to claim 2, characterized in that: The product is a kit or a reagent.

4. A product for detecting Brucella by RT-qPCR, characterized in that: The invention comprises the RT-qPCR primers for detecting the Brucella 23S rRNA gene according to claim 1.

5. The product according to claim 4, characterized in that The product is a kit or a reagent.

6. Use of the RT-qPCR primer for detecting Brucella 23S rRNA gene as claimed in claim 1 or the product as claimed in claim 4 or 5 in RT-qPCR detection of Brucella for non-disease diagnosis purposes.

7. A RT-qPCR detection method for Brucella for non-disease diagnosis purposes, characterized in that, The following steps are involved: Extract the total nucleic acid of the sample to be tested, and reverse transcribe to obtain cDNA; Using the cDNA as a template, qPCR amplification is performed using the RT-qPCR primers for detecting the Brucella 23S rRNA gene according to claim 1 to obtain the Ct value of the sample to be tested, and the Ct value of the sample to be tested is substituted into the standard curve equation to calculate the content of Brucella in the sample to be tested.

8. The RT-qPCR detection method according to claim 7, characterized in that: The reaction system of the qPCR amplification includes: 2×One Step SYBR Green Mix 10µL, One Step SYBR Green Enzyme Mix 1µL, 50×ROX Reference Dye II 0.4µL, 10μM upstream and downstream primers 0.4µL each, template 1µL and RNase-free water 6.5µL.

9. The RT-qPCR detection method according to claim 7, characterized in that: The reaction conditions of the qPCR amplification include: 95°C 30s; cycle reaction 95°C 10s, 53°C 30s, a total of 35 cycles; melting curve reaction 95°C 15s, 60°C 60s, 95°C 15s.

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