CN116590439A - RT-PCR detection primer for group B streptococcus 16S rRNA, probe, kit and application thereof - Google Patents
RT-PCR detection primer for group B streptococcus 16S rRNA, probe, kit and application thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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Abstract
The application discloses a group of RT-PCR detection primers for group B streptococcus 16S rRNA, probes, a kit and application thereof, and relates to the technical field of RT-PCR detection. Comprising an upstream and a downstream primer pairs GBS-16s-F and GBS-16s-R for amplifying and detecting GBS 16s rRNA, and an internal reference primer pair interor-F and interor-R, wherein corresponding probes comprise GBS 16s rRNA detection probes GBS-16s-P and an internal reference probe interor-P. The kit provided by the application can realize the specificity and high-sensitivity detection of the sample RNA, is used for clinical research, and can be used for screening and preventing the group B streptococcus.
Description
Technical Field
The application relates to the technical field of RT-PCR detection, in particular to a group of RT-PCR detection primers for 16S rRNA of streptococcus B, probes, a kit and application thereof.
Background
Group B Streptococcus (group B streptococcus, GBS), also known as Streptococcus agalactiae, is an aerobic gram-positive Streptococcus. The group B streptococcus normally colonizes the vagina and rectum, is a conditional pathogenic bacterium, is colonized in the genital tract of a parturient, can be vertically transmitted to a neonate during delivery, has extremely high mortality rate of neonatal septicemia, meningitis, pneumonia and other diseases caused by neonatal GBS infection, and can cause nervous system sequelae. Currently, GBS has been demonstrated to be one of the major pathogens of perinatal maternal and infant infections, occupying a non-negligible position in perinatal medicine, and it is also the most common cause of sepsis and meningitis in infants. In 2010, the United states disease prevention center formulated the perinatal GBS prevention guidelines, suggesting that pregnant women conduct GBS screening between 35 and 37 weeks of gestation.
At present, the detection methods for the group B streptococcus are more, and the common detection methods are as follows: bacterial culture, colloidal gold detection antibody method, PCR detection of DNA, etc. The bacterial culture method is complicated to operate, requires a long time, usually 24-72 hours, and cannot meet clinical requirements. Colloidal gold methods are generally qualitative or semi-quantitative, and have great limitations in sensitivity and accuracy. Along with the continuous progress of the nucleic acid fluorescent labeling technology, the traditional PCR technology and the spectral technology develop a more sensitive, specific and accurate nucleic acid detection technology, namely a fluorescent quantitative PCR technology. The method has the advantages of accurate detection result, high repeatability and low pollution rate, and the auxiliary detection can be carried out by the fluorescent quantitative PCR technology in clinical diagnosis of the group B streptococcus. However, the existing fluorescent quantitative PCR technology still has the defects of false positive, low accuracy and stability, low sensitivity and the like, and limits the clinical application of the technology. In addition, the existing PCR method generally detects GBS DNA, and the DNA contains some apoptotic cells and dead pathogens, so that the actual condition of pregnant women or newborns infected with group B streptococcus cannot be accurately reflected, sometimes false positive results can occur, clinical medication cannot be well guided, and the clinical application of the PCR method is limited.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a group of RT-PCR detection primers for the 16S rRNA of the group B streptococcus, probes, a kit and application thereof, and by detecting the RNA of the group B streptococcus, the primer can be used as an experimental index for early diagnosis, can reflect the active propagation condition of the group B streptococcus in vivo, has more clinical value, provides a powerful experimental basis for guiding a clinician to treat patients, and solves the technical problem that the current technology cannot accurately reflect the real condition of the group B streptococcus infected by pregnant women or newborns because the DNA contains a plurality of apoptotic cells and dead pathogens.
The technical scheme adopted by the application is as follows:
RT-PCR detection primers for detecting the rRNA of the streptococcus B16S comprise an upstream primer pair GBS-16S-F and a downstream primer pair GBS-16S-R for amplifying and detecting the rRNA of the GBS 16S, and an internal reference primer pair interor-F and interor-R, wherein the nucleotide sequences of the GBS-16S-F, GBS-16S-R, interior-F, interior-R are respectively shown as SEQ ID NO. 1-SEQ ID NO. 4 in sequence.
The specific sequence is as follows:
GBS-16S-F：5’-TTGACATCCTTCTGACCGGC-3’；
GBS-16S-R：5’-AGTCTCGCTAGAGTGCCCAAC-3’；
Interior-F：5’-CAGAGGCAACGACAGGGTAA-3’；
Interior-R：5’-TCTACTTCAGGAAGGCGATGT-3’。
the probes corresponding to the RT-PCR detection primers of the group B streptococcus 16S rRNA comprise GBS 16S-P and an internal reference probe, wherein the GBS 16S-P is shown as SEQ ID NO. 5, and the internal probe is shown as SEQ ID NO. 6.
The specific sequence is as follows:
GBS-16s-P：5’-CTCTTCGGAGCAGAAGTGACAG-3’；
interior-P：5’-TAGCAACTTTCTTGACAGTTCC-3’；
preferably, the nucleotide sequences of GBS-16s-P and integror-P are respectively marked with a fluorescence report group at the 5 'end and a fluorescence quenching group MGB at the 3' end; wherein the fluorescent reporter group is selected from any one of FAM, HEX, VIC, ROX, TET, JOE or CY3, and the nucleotide sequences of GBS-16s-P and integror-P are different in 5' -end fluorescent reporter group
The quenching group of the probe provided by the application adopts a non-fluorescence quenching group NFQ, and the NFQ does not generate fluorescence, so that the intensity of background signals can be greatly reduced, and the detection accuracy is improved; in addition, the probe is connected with a modification group MGB, the MGB is a minor groove binder, the melting point temperature (Tm) value of the probe can be improved by about 10 ℃, and in order to obtain the same Tm value, the MGB probe can be designed to be shorter than a common TaqMan probe, so that the synthesis cost is reduced, and the success rate of probe design is greatly improved.
Further, the 5' -end of the nucleotide sequence of GBS-16s-P is marked with a HEX fluorescent reporter group; and the 5' -end of the nucleotide sequence of the interer-P is marked with a FAM fluorescent reporter group.
A kit for RT-PCR of group B streptococcus 16S rRNA comprises the primer and any one of the probes.
More preferably, the molar concentration ratio of the primer pair GBS-16s-F to GBS-16s-R to the detection probe GBS-16s-P is 1:1:0.2 to 1; the molar concentration ratio of primer pair interier-F to interier-R to internal reference probe interier-P is 1:1:0.2 to 1.
More preferably, the kit further comprises a PCR reaction solution, a positive control solution and a negative control solution; the positive control solution is a plasmid solution containing target fragments; the negative control solution is a solution without group B streptococcus, and is RNase-free water or normal saline; the components of the PCR reaction liquid are as follows: 8-12 mu L of premix system of RT-PCR fluorescent probe method; the molar ratio of the primer pair for GBS 16s rRNA amplification to the GBS 16s rRNA detection probe is as follows: GBS-16s-F: GBS-16s-R: GBS-16 s-p=1: 1:0.2 to 1; the molar ratio of the primer pair amplified by the internal standard to the probe detected by the standard is as follows: intrior-F: intlor-R: intrior-P=1: 1:0.2 to 1; RNase-free water was added to 20. Mu.L.
Further, the reaction conditions of the PCR reaction liquid are as follows:
reverse transcription: 55 ℃ for 10 minutes;
denaturation: 95 ℃ for 1 minute;
annealing, extending and fluorescence acquisition: 30 seconds at 60 ℃ and 45 cycles.
More preferably, the method of use comprises the steps of:
s1, extracting sample RNA;
s2, sample adding: respectively and correspondingly adding sample RNA, negative control solution and positive control solution into a PCR reaction tube/plate filled with the PCR reaction solution, wherein the total volume is 20 mu L respectively, so as to obtain a corresponding sample PCR reaction system, a corresponding negative PCR reaction system and a corresponding positive PCR reaction system;
s3, PCR amplification: respectively placing the sample PCR reaction system, the negative PCR reaction system and the positive PCR reaction system obtained in the step S2 on a fluorescent quantitative PCR instrument, setting a reaction program, and carrying out PCR amplification; wherein the fluorescence detection channel is selected as: selecting HEX channel to detect group B streptococcus, selecting FAM channel to detect internal standard;
s4, judging whether the group B streptococcus is infected or not according to a fluorescence curve after the PCR amplification reaction is finished.
Use of a detection kit for RT-PCR of group B streptococcus 16S rRNA for detecting a complex sample comprising one or more impurities of whole blood, stool, urine, leucorrhea, mucin, human genomic DNA, miconazole suppository, clotrimazole suppository, nystatin ointment, easy-to-stop.
In summary, compared with the prior art, the application has the following advantages and beneficial effects:
1. the application can be used as an experimental index for early diagnosis by detecting the RNA of the group B streptococcus, can reflect the condition of active propagation of the group B streptococcus in vivo at the same time, has more clinical value, and provides a powerful experimental basis for guiding clinicians to treat patients;
2. the kit provided by the application can realize the specificity and high-sensitivity detection of the sample RNA, is used for clinical research, and can be used for screening and preventing the group B streptococcus.
Drawings
FIG. 1 is an amplification curve of a positive reference detected by a group B streptococcus RT-PCR fluorescent probe method detection kit provided in example 2 of the present application;
FIG. 2 is an amplification curve of a negative reference sample detected by the group B streptococcus PCR fluorescent probe method nuclear detection kit provided in example 2 of the present application;
FIG. 3 is an amplification curve of gradient concentration group B streptococcus;
FIG. 4 is a linear relationship of Ct value to logarithm of bacterial concentration;
FIG. 5 is a plot of the amplification of the first reagent lot in the precision test provided in example 2 of the present application;
FIG. 6 is a plot of the amplification of the precision detection in a second reagent lot provided in example 2 of the present application;
FIG. 7 is a plot of the amplification of the third reagent lot in accordance with example 2 of the present application.
Detailed Description
The advantages and various effects of the present application will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the application, not to limit the application.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, 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 application belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
The present application will be described in detail with reference to examples and experimental data.
Example 1
1. Designing a primer and a probe:
in this example, 4 sets of GBS amplification primer pairs, GBS detection probes, internal reference primer pairs and internal reference probes were designed, the nucleotide sequences are shown in Table 1, and the detection conditions of different primers and probes on complex samples were studied.
TABLE 1 nucleotide sequences of primers and probes
2. The preparation steps of the kit are as follows:
the detection kit comprises a primer and a probe for GBS 16S rRNA detection, an RNA extracting solution, a PCR reaction solution, a positive control solution and a negative control solution. Wherein the primer and probe comprise: amplified primer pairs GBS-16S-F and GBS-16S-R, probe GBS-16S-P for target polynucleotide detection, upstream and downstream primer pairs Intoror-F and Intoror-R for internal standard amplification, and probe Intoror-P for internal standard detection.
And HEX fluorescent reporter groups are marked at the 5 'end of the nucleotide sequence of the GBS detection probe GBS-16S-P, non-fluorescent quenching groups are marked at the 3' end, and a modification group MGB is connected. Wherein the fluorescent reporter group may be any one of FAM, TET, JOE, ROX or CY 3.
And the 5 'end of the nucleotide sequence of the probe Interier-P for internal standard detection is marked with FAM fluorescent reporter groups, and the 3' end is marked with non-fluorescent quenching groups and is connected with a modification group MGB. Wherein the fluorescent reporter group may be any one of HEX, TET, JOE, ROX or CY 3.
The concentration ratio of the GBS amplification primer pair to the GBS detection probe is GBS-16S-F: GBS-16S-R: GBS-16S-p=1: 1:0.2 to 1; the concentration ratio of the internal reference primer pair to the internal reference probe is interator-F: integror-R: integror-p=1: 1:0.2 to 1.
The positive control solution is a plasmid solution containing target fragments; the negative control solution is a solution without group B streptococcus, RNase-free water or physiological saline.
Example 2
The embodiment provides the group B streptococcus nucleic acid detection kit of the embodiment 1 for performing an accuracy experiment, and the group B streptococcus PCR fluorescent probe method nucleic acid detection kit provided by the embodiment is used for detecting positive reference RNA, and the specific operation steps are as follows:
1. extracting sample RNA;
2. reagent preparation
And according to the number of the sample to be detected, the positive control liquid and the negative control liquid, the corresponding reaction liquid, the enzyme mixed liquid and the internal standard are proportionally taken, fully and uniformly mixed, and the mixture is subjected to instantaneous centrifugation for later use.
The ratio of the primer pair for GBS 16S rRNA amplification to the GBS 16S rRNA detection probe is as follows: GBS-16S-F: GBS-16S-R: GBS-16S-p=1: 1:0.2 to 1; the ratio of the primer pair amplified by the internal standard to the probe detected by the standard is as follows: intrior-F: intlor-R: intrior-P=1: 1:0.2 to 1. 20. Mu.L of RT-PCR reaction solution was prepared according to the formulation of Table 2.
Table 3-RT-PCR liquid formulation (20. Mu.L)
Wherein, the concentration of the premix system of the PCR-fluorescent probe method is 2×, and the premix system comprises hot start reverse transcriptase, taq DNA polymerase, PCR buffer, dNTPs and MgCl2.
Sample adding: subpackaging the PCR reaction liquid according to the number n of samples to be tested (negative control and positive control are needed), subpackaging the PCR reaction liquid into a PCR reaction tube according to 15-19 mu L/tube, and transferring the PCR reaction tube into a sample treatment area; respectively and correspondingly adding sample RNA, negative control solution and positive control solution into a PCR reaction tube/plate filled with the PCR reaction solution, wherein the total volume is 20 mu L respectively, so as to obtain a corresponding sample PCR reaction system, a corresponding negative PCR reaction system and a corresponding positive PCR reaction system; after short centrifugation for several seconds, all reagents are concentrated to the bottom of the reaction tube, the tube cover is covered or the membrane is sealed, and then the reaction tube is transferred into a nucleic acid amplification area for PCR reaction.
And (3) PCR amplification: and (3) respectively placing the sample PCR reaction system, the negative PCR reaction system and the positive PCR reaction system obtained in the step (3) on a fluorescent quantitative PCR instrument, setting positive control, negative control and unknown samples according to corresponding names, and setting sample names and detection target names.
(1) Fluorescence detection channel selection: selecting HEX channels to detect GBS; FAMC channel detection internal standard is selected.
(2) The reaction procedure was set up as follows:
reverse transcription: 55 ℃ for 10 minutes;
denaturation: 95 ℃ for 1 minute;
annealing, extending and fluorescence acquisition: 30 seconds at 60 ℃ and 45 cycles.
And (3) result judgment: after the PCR amplification reaction is finished, manually adjusting the starting value, the ending value and the threshold line value of the base line for analysis, recording the result of a sample Ct (namely, a cycle number value which is experienced when a fluorescent signal in a PCR reaction tube reaches a set threshold value), and judging whether the group B streptococcus is infected according to the Ct value. The criteria are shown in table 3:
TABLE 3 result criterion
If the sample GBS (HEX channel) has an amplification curve, the sample is positive to the group B streptococcus; if the sample GBS (FAM channel) has no amplification curve and the internal standard (FAM channel) has an amplification curve, the sample GBS is negative to the group B streptococcus; if the amplification curve is not available, the detection result is invalid.
Detection result: the kit detects 8 cases of yin-yang reference products, and the coincidence rate of the yin-yang reference products is 100%. The detection results are shown in table 4 and fig. 1 and 2:
TABLE 4 accuracy test results
| Positive reference | Results | Negative reference | Results |
| P1 | + | N1 | - |
| P2 | + | N2 | - |
| P3 | + | N3 | - |
| P4 | + | N4 | - |
| P5 | + | N5 | - |
| P6 | + | N6 | - |
| P7 | + | N7 | - |
| P8 | + | N8 | - |
FIG. 1 shows an amplification curve of a positive reference for detection by a kit; FIG. 2 shows the amplification curves of the negative reference for the kit detection. As can be seen from fig. 1 and 2, the coincidence rate of 8 reference cases each of negative and positive is 100%.
The amplification curves of 8 positive references of the group B streptococcus are shown, and the amplification curves of 8 references are S-shaped, ct values are provided, ct is larger than 38,8 references are positive, and the cross reaction rate=0 of the 8 positive references indicates that the detection accuracy of the group B streptococcus PCR fluorescent probe method nucleic acid detection kit is higher.
In this example, the analysis performance experiment of the group B streptococcus PCR fluorescent probe nucleic acid detection kit was also evaluated, and RNA extracted from the standard strain was subjected to gradient dilution, and Ct and the logarithm of the bacterial concentration (lg) showed a good linear relationship. The linear regression equation was y= -3.424x+44.198, and the correlation coefficient was 0.991. Each sample was repeated three times. The specific detection results are shown in fig. 3 and 4.
Further, the embodiment also comprises a precision experiment of the nucleic acid detection kit by the group B streptococcus RT-PCR fluorescent probe method, three batches of kits (first, second and third) are respectively used for detecting two samples with low concentration, and each sample is respectively used for detecting 8 repetitions. Specific detection results are shown in the tables 5 to 7 and the summary table 8, and FIGS. 5, 6 and 7 show the first batch of the reagent batch in-precision detection amplification curve, the second batch of the reagent batch in-precision detection amplification curve and the third batch of the reagent batch in-precision detection amplification curve, respectively. The precision experiment shows that the nucleic acid detection kit for the group B streptococcus PCR fluorescent probe method has good in-batch and inter-batch repeatability, and the variation coefficient of the Ct value of the detection result is less than 5%.
TABLE 5 statistics of results of precision measurements in first reagent lot
| Sample number | High (Ct value) | Low (Ct value) |
| 1 | 20.48 | 30.78 |
| 2 | 20.41 | 30.58 |
| 3 | 20.32 | 30.29 |
| 4 | 20.46 | 30.52 |
| 5 | 20.23 | 30.72 |
| 6 | 20.28 | 30.81 |
| 7 | 20.26 | 30.18 |
| 8 | 20.18 | 30.30 |
| Average value of | 20.32 | 30.52 |
| Standard deviation of | 0.11 | 0.24 |
| Coefficient of variation (%) | 0.54 | 0.79 |
TABLE 6 statistics of results of precision measurements in first reagent lot
TABLE 7 statistics of results of precision measurements in third batch of reagents
| Sample number | High (Ct value) | Low (Ct value) |
| 17 | 21.60 | 30.93 |
| 18 | 21.02 | 30.98 |
| 19 | 20.79 | 30.84 |
| 20 | 21.05 | 30.97 |
| 21 | 20.74 | 30.86 |
| 22 | 21.01 | 30.65 |
| 23 | 20.57 | 30.40 |
| 24 | 20.82 | 30.57 |
| Average value of | 20.95 | 30.78 |
| Standard deviation of | 0.31 | 0.21 |
| Coefficient of variation (%) | 1.48 | 0.69 |
TABLE 8 statistics of precision between lots
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
Claims (10)
- The RT-PCR detection primer for detecting the 16S rRNA of the streptococcus B is characterized by comprising an upstream primer pair GBS-16S-F and a downstream primer pair GBS-16S-R for amplifying and detecting the GBS 16S rRNA, wherein the nucleotide sequences of the internal primer pair interor-F and interor-R are respectively shown as SEQ ID NO. 1-SEQ ID NO. 4 in sequence.
- 2. The probe corresponding to the RT-PCR detection primer for the group B streptococcus 16S rRNA, which is disclosed in claim 1, comprises a GBS 16S rRNA detection probe GBS-16S-P and an internal reference probe interer-P, wherein the GBS-16S-P is shown as SEQ ID NO. 5, and the interer-P is shown as SEQ ID NO. 6.
- 3. The probe corresponding to the RT-PCR detection primer for the group B streptococcus 16S rRNA according to claim 2, wherein the nucleotide sequences of GBS-16S-P and interier-P are respectively marked with a fluorescence reporting group at the 5 'end and a fluorescence quenching group MGB at the 3' end; wherein the fluorescent reporter group is selected from any one of FAM, HEX, VIC, ROX, TET, JOE or CY3, and the nucleotide sequences of GBS-16s-P and interer-P are different in 5' -end fluorescent reporter group.
- 4. The probe corresponding to the RT-PCR detection primer for group B streptococcus 16S rRNA according to claim 3, wherein the 5' -end of the nucleotide sequence of GBS-16S-P is marked with HEX fluorescent reporter group; and the 5' -end of the nucleotide sequence of the interer-P is marked with a FAM fluorescent reporter group.
- 5. A kit for RT-PCR of group B streptococcus 16S rRNA comprising the primer of claim 1 and the probe of any one of claims 2 to 4.
- 6. The kit for RT-PCR of group B streptococcus 16S rRNA according to claim 5, wherein the molar ratio of the primer pair GBS-16S-F to GBS-16S-R to the detection probe GBS-16S-P is 1:1:0.2 to 1; the molar concentration ratio of primer pair interier-F to interier-R to internal reference probe interier-P is 1:1:0.2 to 1.
- 7. The kit for RT-PCR of group B streptococcus 16S rRNA according to claim 5, further comprising a PCR reaction solution, a positive control solution and a negative control solution; the positive control solution is a plasmid solution containing target fragments; the negative control solution is a solution without group B streptococcus, and is RNase-free water or normal saline; the components of the PCR reaction liquid are as follows: 8-12 mu L of premix system of RT-PCR fluorescent probe method; the molar ratio of the primer pair for GBS 16s rRNA amplification to the GBS 16s rRNA detection probe is as follows: GBS-16s-F: GBS-16s-R: GBS-16 s-p=1: 1:0.2 to 1; the molar ratio of the primer pair amplified by the internal standard to the probe detected by the standard is as follows: intrior-F: intlor-R: intrior-P=1: 1:0.2 to 1; RNase-free water was added to 20. Mu.L.
- 8. The kit for RT-PCR of group B streptococcus 16S rRNA according to claim 7, wherein the reaction conditions of the PCR reaction solution are:reverse transcription: 55 ℃ for 10 minutes;denaturation: 95 ℃ for 1 minute;annealing, extending and fluorescence acquisition: 30 seconds at 60 ℃ and 45 cycles.
- 9. The kit for RT-PCR of group B streptococcus 16S rRNA of claim 5, wherein the method of use comprises the steps of:s1, extracting sample RNA;s2, sample adding: respectively and correspondingly adding sample RNA, negative control solution and positive control solution into a PCR reaction tube/plate filled with the PCR reaction solution, wherein the total volume is 20 mu L respectively, so as to obtain a corresponding sample PCR reaction system, a corresponding negative PCR reaction system and a corresponding positive PCR reaction system;s3, PCR amplification: respectively placing the sample PCR reaction system, the negative PCR reaction system and the positive PCR reaction system obtained in the step S2 on a fluorescent quantitative PCR instrument, setting a reaction program, and carrying out PCR amplification; wherein the fluorescence detection channel is selected as: selecting HEX channel to detect group B streptococcus, selecting FAM channel to detect internal standard;s4, judging whether the group B streptococcus is infected or not according to a fluorescence curve after the PCR amplification reaction is finished.
- 10. The use of the kit for RT-PCR of group B streptococcus 16S rRNA according to claim 5 for detecting complex samples comprising one or more impurities selected from whole blood, stool, urine, leucorrhea, mucin, human genomic DNA, miconazole suppository, clotrimazole suppository, nystatin ointment, and easy-to-stop.
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| US6004754A (en) * | 1998-01-21 | 1999-12-21 | Becton Dickinson And Company | DNA sequence, related probes and primers for the detection of Streptococcus agalactiae |
| CN107557443A (en) * | 2017-10-27 | 2018-01-09 | 广州赛哲生物科技股份有限公司 | Group B streptococcus fluorescence quantitative PCR detection kit |
| CN111485028A (en) * | 2019-01-29 | 2020-08-04 | 广东美格基因科技有限公司 | Fluorescent quantitative PCR method for detecting tilapia streptococcus agalactiae and corresponding kit |
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