A modified primer probe composition for specific detection of RNA and its application

Abstract

The present invention provides a modified primer probe composition for specific detection of RNA, the modified primer probe composition comprising an upstream primer, a downstream primer and a probe; the upstream primer comprises a 5' end target gene sequence binding region, a complementary structure region, a loop structure region and a 3' end target gene sequence binding region. By inducing the structural conversion of the upstream primer through temperature changes at different stages of PCR, RNA is specifically captured to form cDNA under the condition of coexistence of host DNA and RNA; then cDNA is specifically identified and amplified to avoid interference of the sample's own DNA on amplification; at the same time, the lower limit of RNA detection is reduced and the analysis sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a modified primer probe composition for specifically detecting RNA and an application thereof. Background Art

[0002] In recent years, nucleic acid amplification detection technology has been increasingly used in the field of clinical pathogen detection. Compared with traditional culture detection methods, it has a short detection cycle, high sensitivity and high accuracy, and has become an effective tool for pathogen detection.

[0003] Since DNA is relatively stable, after the pathogens in the test object die, their DNA will still remain in the test object for a long time. In the food field, after the product is sterilized at high temperature, the DNA of the bacteria will still remain on the sample; in the environmental field, after the environment is disinfected by ultraviolet and ozone, the DNA of the bacteria will still remain in the environmental space; in the medical field, after the patient is treated with antibiotics, a large amount of residual DNA of dead bacteria will still remain in the body. When DNA is used as the detection object, the residual DNA of the above pathogens will cause false positive test results, reducing the accuracy of the test results.

[0004] Due to the unstable structure of RNA, RNA is very easy to degrade after the death of the bacteria, and the RNA expression abundance is relatively high. Using RNA as the detection object can improve the accuracy of the test results to a certain extent. Since RNA is transcribed from a DNA template, in prokaryotes, due to their special transcription mechanism, the RNA sequence and the DNA sequence are almost identical except for the difference between single and double strands and bases. This leads to the detection of RNA, and residual DNA will still cause false positive results. In eukaryotes, the RNA nucleic acid sequence is partially similar to the DNA nucleic acid sequence, so residual DNA will also cause false positive results.

[0005] In order to eliminate the interference of DNA on RNA detection, sample DNA is often digested with DNase I or DNAeraser. However, on the one hand, it is difficult for DNase to completely digest DNA, and on the other hand, during the digestion process, RNA is degraded, resulting in damage to RNA integrity, which in turn affects the detection of RNA and increases the missed detection rate.

[0006] In order to improve the detection specificity of RNA, the existing technology uses magnetic bead probes to capture RNA and then detect it. Since the magnetic beads themselves also have the ability to adsorb DNA, it is impossible to completely separate DNA and RNA, and it is impossible to avoid the interference of DNA on RNA detection.

[0007] In order to improve the detection specificity of RNA, the existing technology uses sequence design to make primers bind to shorter target sequences, or introduces mutant bases to improve the specificity of primer-RNA hybridization. However, this method will significantly reduce the detection sensitivity and RT-PCR amplification efficiency.

[0008] Therefore, it is of great significance to develop a primer-probe combination that reduces residual DNA interference and specifically detects RNA molecules in order to improve the accuracy of the test results. Summary of the invention

[0009] In order to solve the above technical problems, the first aspect of the present invention provides a modified primer probe composition for specific detection of RNA, wherein the modified primer probe composition comprises an upstream primer, a downstream primer and a probe sequence; the upstream primer comprises a 5' target gene sequence binding region, a complementary structure region, a loop structure region and a 3' target gene sequence binding region; the 5' target gene sequence binding region comprises 6 to 13 bases, wherein at least 4 bases are complementary to the target gene sequence; the complementary structure region comprises at least 5 complementary base pairs, and its Tm value is 50°C to 55°C; the 3' target gene sequence binding region comprises 3 to 6 bases, wherein at least 3 bases are complementary to the target gene sequence.

[0010] The loop structure region is an artificial sequence and cannot complement the target gene sequence, nor can it form a complementary structure inside. Figure 1 shown.

[0011] The upstream primer of the present invention is relatively long, and the Tm value is 50°C to 55°C, and the Tm value of DNA is usually greater than 55°C. In the process of reverse transcription, when the reverse transcription temperature is 37°C to 50°C, the primer has a hairpin structure. The 5' end target gene sequence binding region and the 3' end target gene sequence binding region of the primer can bind to RNA to form a reverse transcriptase, and form cDNA under the action of RNA polymerase. In this process, because the temperature is lower than the Tm value of DNA, the DNA double strand is not melted, the primer cannot bind to DNA, and can only bind to the single-stranded template RNA, specifically capture RNA, and generate cDNA.

[0012] After obtaining cDNA, PCR amplification is performed. PCR amplification includes denaturation, annealing, extension and amplification. At this stage, the primers, cDNA and DNA are denatured and melted, and the secondary structure is opened to form a linear single-stranded structure. The linear single-stranded upstream primer is only 3 to 6 bases complementary to the DNA template. Because the binding force between the linear single-stranded primer and the DNA template is too weak, it cannot be complementary to the DNA template at a higher annealing and extension temperature, and cannot be extended and amplified with the host DNA as a template, and the reaction is terminated. The linear single-stranded upstream primer has more complementary bases with the cDNA template, and can be complementary to the cDNA template, and can only be amplified with the cDNA template. Therefore, the primer can specifically capture the cDNA and avoid the interference of the host DNA in the amplification.

[0013] As a preferred embodiment, the fluorescent group labeled at the 5' end of the probe is one or more of FAM, VIC, TET, CAL, Gold 540, JOE, HEX, TAMRA, ROX, CY3 or CY5; the quenching group labeled at the 3' end is one or more of MGB, DABCYL, BHQ-1, BHQ-2, BHQ-3, ECLIPE.

[0014] As a preferred embodiment, the modified primer-probe composition is a primer-probe composition for detecting Group B Streptococcus RNA, the upstream primer sequence is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3.

[0015] As a preferred embodiment, the modified primer-probe composition is a primer-probe composition for detecting gonococcal RNA, the upstream primer sequence is shown in SEQ ID NO.4, the downstream primer sequence is shown in SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6.

[0016] As a preferred embodiment, the modified primer-probe composition is a primer-probe composition for detecting Ureaplasma urealyticum RNA, the upstream primer sequence is shown in SEQ ID NO.7, the downstream primer sequence is shown in SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.9.

[0017] The second aspect of the present invention provides a method for specifically detecting RNA, comprising the following steps:

[0018] Step S1: preparing an amplification reaction system solution, the amplification reaction system solution comprising an amplification buffer, an enzyme reaction solution, a sample to be tested, and a modified primer-probe composition, wherein the modified primer-probe composition comprises an upstream primer, a downstream primer, and a probe sequence; the upstream primer comprises a 5' target gene sequence binding region, a complementary structure region, a loop structure region, and a 3' target gene sequence binding region; the 5' target gene sequence binding region comprises 6 to 13 bases, of which at least 4 bases are complementary to the target gene sequence; the complementary structure region comprises at least 5 complementary base pairs, and its Tm value is 50°C to 55°C; the 3' target gene sequence binding region comprises 3 to 6 bases, of which at least 3 bases are complementary to the target gene sequence;

[0019] Step S2: reverse transcription, the reverse transcription temperature is lower than the Tm value of the upstream primer complementary structure region, the upstream primer includes at least one hairpin structure, the 5' end target gene sequence binding region and the 3' end target gene sequence binding region of the upstream primer bind to RNA to form a reverse transcriptase, and reverse transcription forms cDNA;

[0020] Step S3: denaturation, the denaturation temperature is higher than the Tm value of DNA, the primers, cDNA and DNA are denatured and melted to form a single-stranded structure;

[0021] Step S4: annealing, extension, and amplification. The annealing, extension, and amplification temperature is lower than the Tm value of DNA and higher than the Tm value of the complementary structure region of the upstream primer. The upstream primer of the single-stranded structure complementarily binds to the cDNA, and the cDNA is extended and amplified. After amplification, the fluorescence signal is collected.

[0022] As a preferred embodiment, the enzyme reaction solution includes polymerase and reverse transcriptase.

[0023] As a preferred embodiment, the reverse transcription temperature is 40°C to 49°C.

[0024] As a preferred embodiment, the denaturation temperature is 90°C to 100°C.

[0025] As a preferred embodiment, the annealing, extension and amplification temperatures are 56°C to 65°C.

[0026] In the third aspect of the present invention, a kit for specific detection of RNA is provided, the kit comprises an amplification reaction system solution, the amplification reaction system solution comprises a modified primer probe composition; the modified primer probe composition comprises an upstream primer, a downstream primer and a probe; the upstream primer comprises a 5' end target gene sequence binding region, a complementary structure region, a loop structure region and a 3' end target gene sequence binding region; the 5' end target gene sequence binding region comprises 6 to 13 bases, at least 4 of which are complementary to the target gene sequence; the complementary structure region comprises at least 5 complementary base pairs, and its Tm value is 50°C to 55°C; the 3' target sequence binding region is 3 to 6 bases, at least 3 of which are complementary to the target gene sequence. The loop structure region is an artificial sequence, which cannot be complementary to the target gene sequence, and cannot form a complementary structure inside.

[0027] As a preferred embodiment, the kit is a kit for detecting Group B Streptococcus RNA, the upstream primer sequence is shown as SEQ ID NO.1, the downstream primer sequence is shown as SEQ ID NO.2, and the probe sequence is shown as SEQ ID NO.3.

[0028] As a preferred embodiment, the kit is a kit for detecting gonococcal RNA, the upstream primer sequence is shown as SEQ ID NO.4, the downstream primer sequence is shown as SEQ ID NO.5, and the probe sequence is shown as SEQ ID NO.6.

[0029] As a preferred embodiment, the kit is a kit for detecting Ureaplasma urealyticum RNA, the upstream primer sequence is shown as SEQ ID NO.7, the downstream primer sequence is shown as SEQ ID NO.8, and the probe sequence is shown as SEQ ID NO.9.

[0030] As a preferred embodiment, the amplification reaction system solution further includes an amplification buffer and an enzyme reaction solution; the enzyme reaction solution includes a polymerase and a reverse transcriptase.

[0031] The fourth aspect of the present invention provides the use of the modified primer-probe composition in a kit for specifically detecting RNA.

[0032] As a preferred embodiment, the application steps include reverse transcription, denaturation, annealing, extension and amplification; the reverse transcription temperature is lower than the Tm value of the upstream primer complementary structure region; the annealing, extension and amplification temperatures are lower than the Tm value of DNA and higher than the Tm value of the upstream primer complementary structure region.

[0033] Through the above technical solutions, the present invention produces the following technical effects:

[0034] (1) The present invention specifically detects RNA through dual technology. In the RNA reverse transcription stage, the reverse transcription temperature is lower than the Tm value of the complementary structure region of the modified upstream primer, and the primer has a hairpin structure. The target sequence binding region at the 5' end of the primer and the target sequence binding region at the 3' end can only bind to single-stranded RNA to form a reverse transcriptase, and cDNA is formed under the action of RNA polymerase. In the denaturation, annealing, extension, and amplification cycle stages, the temperature is higher than the Tm value of the complementary structure region of the upstream primer, and the modified primer presents a linear single-stranded structure, which specifically recognizes cDNA, binds to it, and amplifies it, thereby achieving specific capture of cDNA under conditions where DNA and RNA coexist, avoiding interference of DNA in RNA amplification.

[0035] (2) The technical solution of the present invention can reduce the lower limit of RNA detection and improve the analysis sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the upstream primer sequence structure, where 1 is the 5' end target gene sequence binding region; 2 is the complementary structure region; 3 is the loop structure region; and 4 is the 3' end target gene sequence binding region. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] This method has no special requirements for the RNA extraction method, as long as it complies with the conventional RNA extraction method in the art.

[0039] Example 1: Detection of Group B Streptococcus (GBS) in a sample using the technical solution of the present invention

[0040] Group B Streptococcus (GBS) is a Gram-positive bacterium that can parasitize the reproductive tract of pregnant women and cause infection in infants. Example 1 The gene of Group B Streptococcus was used as the detection object to verify the effect of the present invention.

[0041] 1. Sample preparation

[0042] The samples include RNA positive samples, DNA positive samples, and negative controls.

[0043] RNA positive samples: GBS culture positive bacteria were prepared into a bacterial concentration of 10 3 cfu / ml, 10 2cfu / ml, 50 cfu / ml, and 5 cfu / ml bacterial solutions were used as RNA sample sources. A commercial RNA extraction kit was used to extract positive bacterial concentrations of 10 3 cfu / ml, 10 2 cfu / ml, 50 cfu / ml, and 5 cfu / ml of RNA were correspondingly made into RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, and RNA positive sample 4.

[0044] DNA positive sample: GBS 16sDNA sequence plasmid was used as DNA positive sample, and the plasmid concentration was 10 8 The DNA positive sample was 1, and the plasmid concentration was 10 copies / ml. 6 copies / ml of DNA positive sample 2.

[0045] Physiological saline was used as negative control.

[0046] 2. Primer and probe design

[0047] The GBS 16s RNA sequence was downloaded from the bioinformatics website. Primers and probes were designed based on the GBS conserved sequence through bioinformatics specific comparison analysis. The specific detection sequences are shown in the following table.

[0048] Table 1: Primer and probe sequences

[0049]

[0050] 3. Amplification reaction system solution configuration

[0051] The solution configuration of the one-step amplification reaction system is shown in the table below.

[0052] Table 2: Amplification reaction system solution

[0053]

[0054] Note: * indicates that the samples were added to the amplification reaction system solution using the above primers for RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, RNA positive sample 4, DNA positive sample 1, DNA positive sample 2, and negative control, and tested using qRT-PCR. Each sample was tested three times in parallel.

[0055] 4. Reaction procedure

[0056] The prepared amplification reaction system solution was mixed and centrifuged, and amplified using an ABI 7500 PCR amplification instrument according to the reaction program. The reaction program is shown in the table below.

[0057] Table 3: Reaction Procedure

[0058]

[0059] 5. Test results and analysis

[0060] The test results are shown in the table below. When the bacterial concentration is as low as 5 cfu / ml, the technical solution of the present invention can still effectively detect RNA, and the Ct value is small.

[0061] The DNA concentration in the sample was as high as 10 8 When the concentration of RNA was 0.14475 μg / ml, DNA was still not detected by the scheme of the present invention, and the Ct value was small, indicating that the scheme can effectively identify and distinguish RNA and DNA, avoid the interference of DNA on RNA detection, and specifically detect RNA.

[0062] The difference in Ct values ​​of the three parallel tests was small, indicating that the technical solution of the present invention has high stability.

[0063] Table 4: Experimental results of Example 1

[0064]

[0065] In this application, Undet means not detected.

[0066] Comparative Example 1: Detection of Group B Streptococcus (GBS) in samples using conventional PCR technology

[0067] 1. Sample preparation

[0068] Same as Example 1.

[0069] 2. Primer and probe design

[0070] The GBS 16s RNA sequence was downloaded from the bioinformatics website. Primers and probes were designed based on the GBS conserved sequence through bioinformatics specific comparison analysis. The specific detection sequences are shown in the following table.

[0071] Table 5: Primer and probe sequences

[0072]

[0073] 3. Preparation of amplification reaction system solution

[0074] Conventional PCR amplification reaction system solutions are shown in the table below.

[0075] Table 6: Conventional PCR amplification reaction system solution

[0076]

[0077] Note: * indicates that the samples were added to the amplification reaction system solution using the above primers for RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, RNA positive sample 4, DNA positive sample 1, DNA positive sample 2, and negative control, and tested using qRT-PCR. Each sample was tested three times in parallel.

[0078] 4. Reaction procedure

[0079] The prepared amplification reaction system solution was mixed and centrifuged, and amplified using an ABI 7500 PCR amplification instrument according to the reaction program. The reaction program is shown in the table below.

[0080] Table 7: General PCR reaction procedure

[0081]

[0082] 5. Test results and analysis The test results are shown in the table below.

[0083] Comparative Example 1 and Example 1 were compared and analyzed. The results showed that when the bacterial concentration was 5 cfu / ml to 50 cfu / ml, the conventional PCR technical solution did not effectively detect RNA. When the bacterial concentration was 5 cfu / ml to 50 cfu / ml, the solution of the present invention could effectively and stably detect RNA, indicating that the technical solution of the present invention can reduce the lower limit of RNA detection and improve the analysis sensitivity.

[0084] When the DNA concentration in the sample is 10 6 When the concentration of DNA in the sample is as high as 10 copies / ml, the conventional PCR technology can detect RNA and DNA at the same time. This shows that the conventional PCR technology cannot distinguish between RNA and DNA, DNA interferes with RNA detection, and the specificity of RNA detection is low. 8 When the concentration of RNA was 2.3477 copies / ml, the technical solution of the present invention was used to detect RNA, but DNA was still not detected, and the Ct value was small. This shows that the technical solution of the present invention can effectively identify and distinguish RNA and DNA, avoid DNA in the sample interfering with RNA detection, and specifically detect RNA.

[0085] Table 8: Comparative Example 1 Test Results

[0086]

[0087] Example 2: Detection of gonococci (NG) in samples using the technical solution of the present invention

[0088] Gonorrhea (NG) is a Gram-negative bacterium that can parasitize the human body and cause purulent infection of the urogenital system. It is one of the common sexually transmitted diseases. Example 2 uses gonococcal genes as the detection object to verify the effect of the present invention.

[0089] 1. Sample preparation

[0090] The samples include RNA positive samples, DNA positive samples, and negative controls.

[0091] RNA positive samples: The bacterial cells positive for clinical NG culture were made into a bacterial concentration of 10 3 cfu / ml, 10 2 cfu / ml, 50 cfu / ml, and 5 cfu / ml bacterial solutions were used as RNA sample sources. A commercial RNA extraction kit was used to extract positive bacterial concentrations of 10 3 cfu / ml, 10 2 cfu / ml, 50 cfu / ml, and 5 cfu / ml of RNA were correspondingly made into RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, and RNA positive sample 4.

[0092] DNA positive sample: NG 16sDNA sequence plasmid was used as DNA positive sample, and the plasmid concentration was 10 8 The DNA positive sample was 1, and the plasmid concentration was 10 copies / ml. 6 copies / ml of DNA positive sample 2.

[0093] Physiological saline was used as negative control.

[0094] 2. Primer and probe design

[0095] Download the NG 16s RNA sequence from the bioinformatics website, perform specific comparison analysis using bioinformatics, and design primers and probes based on the NG conservative sequence. The specific detection sequence is as follows:

[0096] Table 9: Primer and probe sequences

[0097]

[0098] 3. Amplification reaction system solution configuration

[0099] The solution configuration of the one-step amplification reaction system is shown in the table below.

[0100] Table 10: Amplification reaction system solution

[0101]

[0102] Note: * indicates that the samples were added to the amplification reaction system solution using the above primers for RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, RNA positive sample 4, DNA positive sample 1, DNA positive sample 2, and negative control, and tested using qRT-PCR. Each sample was tested three times in parallel.

[0103] 4. Reaction procedure

[0104] The prepared amplification reaction system solution was mixed and centrifuged, and amplified using an ABI 7500 PCR amplification instrument according to the reaction program. The reaction program is shown in the table below.

[0105] Table 11: Reaction Procedure

[0106]

[0107] 5. Test results and analysis

[0108] The test results are shown in the table below. When the bacterial concentration in the sample is as low as 5 cfu / ml, the technical solution of the present invention can still effectively detect RNA, and the Ct value is small.

[0109] The DNA concentration in the sample was as high as 10 8 When the concentration of 500 copies / ml was 2.5, DNA was still not detected by the scheme of the present invention, and the Ct value was small, indicating that the scheme can effectively identify RNA and DNA, avoid the interference of DNA on RNA detection, and specifically detect RNA.

[0110] The difference in Ct values ​​of the three parallel tests was small, indicating that the technical solution of the present invention has high stability.

[0111] Table 12: Experimental results of Example 2

[0112]

[0113] Comparative Example 2: Using conventional PCR technology to detect NG in samples

[0114] 1. Sample preparation

[0115] Same as Example 2.

[0116] 2. Primer and probe design

[0117] Download the NG 16s RNA sequence from the bioinformatics website, perform specific comparison analysis using bioinformatics, and design primers and probes based on the NG conservative sequence. The specific detection sequence is as follows:

[0118] Table 13: Primer and probe sequences

[0119]

[0120] 3. Preparation of amplification reaction system solution

[0121] Same as Example 2.

[0122] Table 14: Conventional PCR amplification reaction system solution

[0123]

[0124] Note: * indicates that the samples were added to the amplification reaction system solution using the above primers for RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, RNA positive sample 4, DNA positive sample 1, DNA positive sample 2, and negative control, and tested using qRT-PCR. Each sample was tested three times in parallel.

[0125] 4. Reaction procedure

[0126] The prepared amplification reaction system solution was mixed and centrifuged, and amplified using an ABI 7500 PCR amplification instrument according to the reaction program. The reaction program is shown in the table below.

[0127] Table 15: General PCR reaction procedure

[0128]

[0129] 5. Test results and analysis

[0130] The test results are shown in the table below.

[0131] Comparative Example 2 and Example 2 were compared and analyzed. The results showed that when the bacterial concentration was 5 cfu / ml to 50 cfu / ml, the conventional PCR technical solution did not effectively detect RNA. When the strain concentration was 5 cfu / ml or 50 cfu / ml, the solution of the present invention could effectively and stably detect RNA, indicating that the technical solution of the present invention can reduce the lower limit of RNA detection and improve the analysis sensitivity.

[0132] When the DNA concentration in the sample is 10 6 When the concentration of DNA in the sample is as high as 10 copies / ml, the conventional PCR technology can detect RNA and DNA at the same time. This shows that the conventional PCR technology cannot distinguish between RNA and DNA, DNA interferes with RNA detection, and the specificity of RNA detection is low. 8 When the concentration of RNA was 2.3477 copies / ml, the technical solution of the present invention was used to detect RNA, but DNA was still not detected, and the Ct value was small. This shows that the technical solution of the present invention can effectively identify and distinguish RNA and DNA, avoid DNA in the sample interfering with RNA detection, and specifically detect RNA.

[0133] Table 16: Comparative Example 2 Test Results

[0134]

[0135] Example 3: Detection of Ureaplasma urealyticum (UU) in a sample using the technical solution of the present invention

[0136] Ureaplasma urealyticum (UU) is a type of Ureaplasma urealyticum, mainly inhabits the urogenital tract of the human body, and is one of the important pathogens of sexually transmitted diseases. Example 3 uses the Ureaplasma urealyticum gene as the detection object to verify the effect of the present invention.

[0137] 1. Sample preparation

[0138] The samples include RNA positive samples, DNA positive samples, and negative controls.

[0139] RNA positive samples: The bacterial cells positive for clinical UU culture were made into a bacterial concentration of 10 3 cfu / ml, 10 2 cfu / ml, 50 cfu / ml, and 5 cfu / ml bacterial solutions were used as RNA sample sources. A commercial RNA extraction kit was used to extract positive bacterial concentrations of 10 3 cfu / ml, 10 2 cfu / ml, 50 cfu / ml, and 5 cfu / ml of RNA were correspondingly made into RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, and RNA positive sample 4.

[0140] DNA positive sample: UU 16sDNA sequence plasmid was used as DNA positive sample, and the plasmid concentration was 10 8 The DNA positive sample was 1, and the plasmid concentration was 10 copies / ml. 6 copies / ml of DNA positive sample 2.

[0141] Physiological saline was used as negative control.

[0142] 2. Primer and probe design

[0143] Download the UU 16s RNA sequence from the bioinformatics website, perform specific comparison analysis using bioinformatics, and design primers and probes based on the UU conservative sequence. The specific detection sequence is as follows:

[0144] Table 17: Primer and probe sequences

[0145]

[0146] 3. Amplification reaction system solution configuration

[0147] The solution configuration of the one-step amplification reaction system is shown in the table below.

[0148] Table 18: Amplification reaction system solution

[0149]

[0150] Note: * indicates that the samples were added to the amplification reaction system solution using the above primers for RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, RNA positive sample 4, DNA positive sample 1, DNA positive sample 2, and negative control, and tested using qRT-PCR. Each sample was tested three times in parallel.

[0151] 4. Reaction procedure

[0152] The prepared amplification reaction system solution was mixed and centrifuged, and amplified using an ABI 7500 PCR amplification instrument according to the reaction program. The reaction program is shown in the table below.

[0153] Table 19: Reaction schedule

[0154]

[0155] 5. Test results and analysis

[0156] The test results are shown in the table below. When the bacterial concentration in the sample is as low as 5 cfu / ml, the technical solution of the present invention can still effectively detect RNA, and the Ct value is small.

[0157] The DNA concentration in the sample was as high as 10 8 When the concentration of RNA was 0.14475 μg / ml, DNA was still not detected by the scheme of the present invention, and the Ct value was small, indicating that the scheme can effectively identify and distinguish RNA and DNA, avoid the interference of DNA on RNA detection, and specifically detect RNA.

[0158] The difference in Ct values ​​of the three parallel tests was small, indicating that the technical solution of the present invention has high stability.

[0159] Table 20: Experimental results of Example 3

[0160]

[0161] Comparative Example 3: Detection of UU in samples using conventional PCR technology

[0162] 1. Sample preparation

[0163] Same as Example 3.

[0164] 2. Primer and probe design

[0165] The UU 16s RNA sequence was downloaded from the bioinformatics website. Through bioinformatics specific comparison analysis, primers and probes were designed according to the UU conserved sequence. The specific detection sequences are shown in the following table.

[0166] Table 21: Primer and probe sequences

[0167]

[0168] 3. Preparation of amplification reaction system solution

[0169] The amplification reaction system solution is shown in the table below.

[0170] Table 22: Conventional PCR amplification reaction system solution

[0171]

[0172] Note: * indicates that the samples were added to the amplification reaction system solution using the above primers for RNA positive sample 1, RNA positive sample 2, RNA positive sample 3, RNA positive sample 4, DNA positive sample 1, DNA positive sample 2, and negative control, and tested using qRT-PCR. Each sample was tested three times in parallel.

[0173] 4. Reaction procedure

[0174] The prepared amplification reaction system solution was mixed and centrifuged, and amplified using an ABI 7500 PCR amplification instrument according to the reaction program. The reaction program is shown in the table below.

[0175] Table 23: General PCR reaction procedure

[0176]

[0177] 5. Test results and analysis

[0178] The test results are shown in the table below.

[0179] Comparative Example 3 and Example 3 were compared and analyzed. The results showed that when the bacterial concentration was 5 cfu / ml, the conventional PCR technical solution did not effectively detect RNA; when the strain concentration was 50 cfu / ml, the conventional PCR technical solution did not stably detect RNA. When the strain concentration was 5 cfu / ml to 50 cfu / ml, the solution of the present invention could effectively and stably detect RNA, indicating that the detection of the technical solution of the present invention can reduce the lower limit of RNA detection and improve the analysis sensitivity.

[0180] When the DNA concentration in the sample is 10 6When the concentration of DNA in the sample is as high as 10 copies / ml, the conventional PCR technology can detect RNA and DNA at the same time. This shows that the conventional PCR technology cannot distinguish between RNA and DNA, DNA interferes with RNA detection, and the specificity of RNA detection is low. 8 When the sample size was 200 copies / ml, the technical solution of the present invention was used to detect RNA, but DNA was still not detected, and the Ct value was small. This shows that the technical solution of the present invention can effectively identify and distinguish RNA and DNA, avoid DNA in the sample interfering with RNA detection, and can specifically detect RNA.

[0181] Table 24: Comparative Example 3 Test Results

[0182]

[0183] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A modified primer-probe composition for specific detection of RNA, characterized in that: The modified primer probe composition comprises an upstream primer, a downstream primer and a probe sequence; the upstream primer comprises a 5' target gene sequence binding region, a complementary structure region, a loop structure region and a 3' target gene sequence binding region; the 5' target gene sequence binding region comprises 6 to 13 bases, at least 4 of which are complementary to the target gene sequence; the complementary structure region comprises at least 5 complementary base pairs, and its Tm value is 50°C to 55°C; the 3' target gene sequence binding region comprises 3 to 6 bases, at least 3 of which are complementary to the target gene sequence.

2. The modified primer probe composition according to claim 1, characterized in that: The modified primer-probe composition is a primer-probe composition for detecting Group B Streptococcus RNA, the upstream primer sequence is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.

3.

3. The modified primer probe composition according to claim 1, characterized in that: The modified primer-probe composition is a primer-probe composition for detecting gonococcal RNA, the upstream primer sequence is shown in SEQ ID NO.4, the downstream primer sequence is shown in SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.

6.

4. The modified primer probe composition according to claim 1, characterized in that: The modified primer-probe composition is a primer-probe composition for detecting Ureaplasma urealyticum RNA, the upstream primer sequence is shown in SEQ ID NO.7, the downstream primer sequence is shown in SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.

9.

5. A kit for specific detection of RNA, characterized in that: The kit comprises a modified primer-probe composition for specifically detecting RNA as described in any one of claims 1 to 4.

6. The kit for specific detection of RNA according to claim 5, characterized in that: The kit also includes an amplification reaction system solution, which also includes an amplification buffer and an enzyme reaction solution; the enzyme reaction solution includes a polymerase and a reverse transcriptase.

7. Use of a modified primer-probe composition for specific RNA detection according to any one of claims 1 to 4 in the preparation of a kit for specific RNA detection.

8. The use according to claim 7, characterized in that: The application steps include reverse transcription, denaturation, annealing, extension and amplification; the reverse transcription temperature is lower than the Tm value of the upstream primer complementary structure region; the annealing, extension and amplification temperatures are lower than the Tm value of DNA and higher than the Tm value of the upstream primer complementary structure region.

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