CN113025733A - Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit - Google Patents

Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit Download PDF

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CN113025733A
CN113025733A CN202110276766.2A CN202110276766A CN113025733A CN 113025733 A CN113025733 A CN 113025733A CN 202110276766 A CN202110276766 A CN 202110276766A CN 113025733 A CN113025733 A CN 113025733A
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streptococcus
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于超计
吴文立
王倩玉
赵立明
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Beijing Huarui Kangyuan Biotechnology Development Co ltd
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Abstract

The invention discloses a novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit, which comprises an amplification reaction MIX, a GBS detection solution, a positive control substance, a negative control substance and a sample releasing agent; the GBS detection solution comprises a reagent against group B streptococcusCAMPSpecific marker primer of gene, specific marker primer of internal reference gene RNAse P and RNase Free ddH2And O. The novel kit for detecting the group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography is characterized in that after nucleic acid is extracted from a collected cell sample, a specific fragment is amplified under the action of Taq DNA polymerase, and then the group B streptococcus nucleic acid is detected by using the colloidal gold immunochromatography, and in addition, the group B streptococcus nucleic acid is detectedIn addition, the quality control tracking of the internal reference gene RNAse P is carried out in the processes of sample collection, nucleic acid extraction and PCR amplification, so that the whole experimental process is ensured to be in a controllable range.

Description

Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit
Technical Field
The invention relates to the technical field of biological detection, in particular to a novel kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography.
Background
Group B streptococcus is a gram-positive coccus, known as streptococcus agalactiae (s.agalactiae), which was early appreciated by the veterinary community because it can cause mastitis in cattle, seriously compromising animal husbandry. Up to 1938, Fry reported for the first time that 3 human cases infected with group B streptococci caused death of postpartum endocarditis, confirming that group B streptococci are also pathogenic in humans. The polysaccharide substance in the cell wall of this bacterium belongs to Group B of the structural classification of antigens, and is also called Group B Streptococcus (GBS).
Group B streptococci are conditionally pathogenic bacteria and are the leading cause of invasive infection in neonates, which is internationally accepted. If pregnant women are infected with group B streptococci, newborns may inhale the infected amniotic fluid at birth or infect group B streptococci through the birth canal. Infection of group B streptococci in newborns is primarily manifested as an early invasive infection, with neonatal sepsis as the major clinical manifestation. Infection of newborn with group B streptococci can lead to septicemia, pneumonia and meningitis, with higher morbidity and mortality, and can also leave long-term pathological conditions such as deafness, impaired vision, developmental disorders and cerebral palsy. Meanwhile, group B streptococci can also cause premature birth, fetal dysplasia (low body weight), premature rupture of the fetal membrane and late stage abortion. Survey statistics over 10 years in the new hafen region of the united states show: the annual incidence of neonatal sepsis is 2.7%, of which about 50% is caused by group B streptococcal infection. Antibiotics (especially penicillin) are effective means for treating group B streptococcal infections, so that group B streptococcal infection screening before delivery of pregnant women is especially necessary to avoid antibiotic abuse and effectively reduce neonatal GBS infection.
In order to reduce the infection of newborns caused by the infection of group B streptococcus, the Chinese medical society, the obstetric scientific society, the labor science group, in "health guidance recommendation before and during pregnancy" (2018 edition), definitely screens GBS as a standby item for high-risk pregnant women, and the optimal detection time is 35-37 weeks. Therefore, an effective and quick pregnant and lying-in woman GBS screening method is established, and the method has important significance for reducing neonatal GBS infection.
The current laboratory detection scheme for group B streptococcal infection mainly comprises a traditional culture method and a fluorescent quantitative PCR method. The culture method, as a gold standard for detecting pathogenic microorganisms, has the remarkable advantage of high specificity, but has a series of defects of long time consumption, high cost, high culture difficulty, low flux, low sensitivity and the like, and is difficult to meet the actual clinical requirements; the fluorescent quantitative PCR method has the advantages of high sensitivity, short time consumption and relatively high flux, but the detection process of the method depends on an expensive qPCR instrument, and the requirements of primary hospitals are difficult to meet.
The nucleic acid colloidal gold immunochromatography technology is characterized in that specific antigens or antibodies are fixed on an NC membrane in a strip shape to form a detection line (T line), an internal reference line (R line) and a quality control line (C line), and a colloidal gold labeled reagent (antibodies or monoclonal antibodies) is adsorbed on a binding pad. After a sample to be detected is extracted by conventional nucleic acid, PCR amplification is carried out by adopting a primer with a specific mark at the 5' end, an amplification product is directly added on a sample pad at one end of a test strip, the amplification product moves forwards through capillary action and interacts with a colloidal gold labeling reagent on a combination pad, and when the amplification product moves to a region of fixed antigen or antibody, a combination of an object to be detected and the colloidal gold labeling reagent is specifically combined with the combination and is intercepted, and the combination is gathered on a detection band to form a visual color development result (as shown in figure 1). The method is widely applied to antigen-antibody detection, but is rarely used in the field of nucleic acid detection.
In the whole experiment process, pollution or other interference factors may appear by sample nucleic acid extraction and PCR amplification, and the traditional colloidal gold only has two detection lines, cannot be provided with reference gene detection, cannot effectively monitor the whole nucleic acid detection process, and may cause false negative results and interference judgment.
The invention aims to establish a kit which can simply, conveniently, quickly, accurately and high-flux detect the GBS infection condition of a pregnant woman in 35-37 weeks by utilizing a PCR (polymerase chain reaction) technology and a colloidal gold immunochromatography technology. The reagent kit is internally provided with an internal reference gene for whole-process quality control, so that false negative result interference can be avoided.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a novel kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography, which can overcome the defects in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit comprises an amplification reaction MIX, a GBS detection solution, a positive control substance, a negative control substance and a sample releasing agent;
the GBS detection solution comprises a specific marker primer aiming at group B streptococcus CAMP genes, a specific marker primer of an internal reference gene RNAse P and RNase Free ddH2O；
The specific marker primer sequence aiming at the group B streptococcus CAMP gene is as follows:
forward primer sequence: 5 '-Bio-CTCTAGTGGCTGGTGCATTGTT-3', as shown in SEQ ID NO.1,
reverse primer sequence: 5 '-TAMARA-GAGTTGTCACTTGATCAGCATGT-3' as shown in SEQ ID NO. 2;
marking biotin at the 5' end of the forward primer; the reverse primer may be labeled at the 5' end with FITC or TAMARA, preferably TAMARA.
The specific marker primer sequence of the internal reference gene RNAse P is as follows:
forward primer sequence: 5 '-Bio-AGATTTGGACCTGCGAGCG-3', as shown in SEQ ID NO.3,
reverse primer sequence: 5 '-DIG-GAGCGGCTGTCTCCACAAGT-3' as shown in SEQ ID NO. 4;
marking biotin at the 5' end of the forward primer; the reverse primer may be labeled at the 5' end with FITC or DIG, preferably DIG.
Preferably, the amplification reaction MIX comprises Taq DNA polymerase, 4 dntps and ions required for PCR amplification reaction solution.
Preferably, the positive control is an inactivated streptococcus group B strain.
Preferably, the negative control is RNase Free ddH2O。
Preferably, the sample-releasing agent is a cell lysate.
Preferably, the concentration of the specific marker primer aiming at the group B streptococcus CAMP gene in the GBS detection solution is 50-400 nM.
Preferably, the concentration of the specific marker primer for group B Streptococcus CAMP gene in the GBS test solution is 200 nM.
Preferably, the concentration of the specific marker primer of the reference gene RNAse P in the GBS detection solution is 50-400 nM.
Preferably, the concentration of the specific marker primer of the internal reference gene RNAse P in the GBS detection solution is 400 nM.
The invention has the beneficial effects that:
(1) fills up the clinical blank: the novel kit for detecting the group B streptococcus nucleic acid by the PCR-colloidal gold immunochromatography can quickly, accurately and sensitively detect the group B streptococcus, and fills the blank field of the detection of the group B streptococcus infection nucleic acid colloidal gold of the pregnant women in the clinical practice;
(2) the clinical diagnosis efficiency is accelerated: the experimental result has good repeatability and high precision; the invention has short detection time period, can finish detection within 70 minutes at the fastest speed, and greatly saves the detection time;
(3) quality control, effectively prevent false negative: the quality control strip is designed for the colloidal gold chromatography test paper, so that the quality of the colloidal gold chromatography process can be monitored, and whether false positive occurs or not and manual operation errors can be monitored; and the internal reference gene is also set to carry out whole-process tracking quality control on sample collection, nucleic acid extraction and PCR amplification, and supervise the whole process;
(4) simple operation, be applicable to basic level hospital: the method can be used for detecting the streptococcus B by only mixing the group B streptococcus nucleic acid detection solution with the template by an operator, the detection instrument only depends on a common PCR instrument, the nucleic acid colloidal gold immunochromatographic test result is visual, the requirements on operators and instruments are extremely low, and the kit is suitable for high-level, medium-level and low-level hospitals and has high clinical popularization.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of the detection principle of nucleic acid colloidal gold immunochromatography.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1
A novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit comprises:
(1) amplification reaction MIX: purchased from Nanjing Novowed Biotechnology Ltd (product number: P211-V10.1), the reaction system comprises Taq DNA polymerase, 4 dNTPs and ions required by various PCR amplification reaction solutions.
(2) GBS detection liquid: comprises specific marker primers aiming at group B streptococcus CAMP genes, specific marker primers of internal reference gene RNAse P and RNase Free ddH2O。
Group B streptococcus amplification primers:
CX-CFB-F:5 '-Bio-CTCTAGTGGCTGGTGCATTGTT-3', shown in SEQ ID NO.1,
CX-CFB-R is 5 '-TAMARA-GAGTTGTCACTTGATCAGCATGT-3', as shown in SEQ ID NO.2,
marking biotin at the 5' end of the forward primer; the reverse primer was labeled TAMARA at the 5' end. The primer concentration is 50-400 nM, preferably 200 nM.
Specific marker primers of internal reference gene RNAse P:
RNAse P-F: 5 '-Bio-AGATTTGGACCTGCGAGCG-3', as shown in SEQ ID NO.3,
RNAse P-R: 5 '-DIG-GAGCGGCTGTCTCCACAAGT-3', as shown in SEQ ID NO.4,
marking biotin at the 5' end of the forward primer; the reverse primer is labeled with DIG at the 5' end. The primer concentration is 50-400 nM, preferably 400 nM.
(3) Positive control: inactivated Streptococcus group B strains (purchased from ATCC).
(4) Negative control: RNase Free ddH2O。
(5) Chromatography strip: and the chromatographic test paper is coated with the colloidal gold particles and the antibody.
(6) Sample releasing agent: cell lysate (purchased from Signosis, USA, goods number: CL-0001)
In this example, the group B Streptococcus CAMP gene sequence (CAMP gene sequence is shown in SEQ ID NO. 5) and the internal reference gene RNAse P gene sequence (RNAse P gene partial sequence is shown in SEQ ID NO. 6) were selected and compared, and a pair of specific primers were designed. Ensures that the two pairs of primers can specifically amplify the group B streptococcus CAMP gene and the RNAse P gene, while other pathogenic bacteria (such as streptococcus pneumoniae, streptococcus pyogenes, streptococcus thermophilus, streptococcus mutans, streptococcus pyogenes, lactobacillus acidophilus, lactobacillus reuteri, staphylococcus epidermidis, escherichia coli DH5 alpha and candida albicans) cannot amplify.
The components of the kit are shown in table 1, and the kit is stored at-20 ℃.
TABLE 1 Components of the kit
Figure BDA0002976963930000061
The method for performing the colloidal gold chromatography detection on the group B streptococcus nucleic acid by using the kit of the embodiment is to use the group B streptococcus DNA as a template to prepare an amplification reaction system for PCR amplification, and simultaneously, an internal reference gene RNAse P performs whole-process quality control on sample collection, nucleic acid extraction and PCR amplification, so that the result controllability of the whole process is ensured, and the detection principle of the nucleic acid colloidal gold immunochromatography is shown in FIG. 1.
The amplification reaction system comprises: sample template, amplification reaction MIX and detection solution.
The amplification procedure is as follows:
95 ℃ for 3min (1 cycle);
95 ℃ 15sec, 58 ℃ 30sec, 72 ℃ 30sec (35 cycles);
5min at 72 ℃ (1 cycle);
4 ℃ infinity (1 cycle).
Further, after the PCR reaction is completed, the following operations are sequentially performed:
a. diluting the amplification product by 10 times with sample diluent, wherein the dilution times can be adjusted according to the product concentration;
b. taking out the chromatographic test strip and placing the chromatographic test strip on a clean horizontal desktop (for use as soon as possible), and paying attention not to touch an NC membrane;
c. and (3) sucking 80 mu L of sample to be detected by a pipette or a dropper, slowly and dropwise adding the sample to be detected on the sample adding hole of the test strip, and judging the result after 2-10 minutes.
The results are analyzed as shown in table 2:
TABLE 2 analysis of the results
Figure BDA0002976963930000071
Example 2
Performance verification of the kit described in example 1
(1) Sample processing
A200. mu.L sample was taken, 100. mu.L sample releasing agent was added, and the mixture was allowed to stand on ice for 10min and centrifuged at 10,000g for 2min by a centrifuge. 100 μ L of the supernatant was taken for further use (long-term storage at-80 ℃).
(2) PCR amplification
PCR amplification reaction solutions (20. mu.L per reaction) were prepared as shown in Table 3
TABLE 3 PCR amplification reaction solution
Components 1 reaction volume
GBS detection liquid 7.5μL
Amplification reaction MIX 12.5μL
Total volume 20μL
The prepared PCR amplification reaction solution was dispensed into each reaction well of 20. mu.L. Adding 5 mul of extracted sample DNA, positive reference substance DNA and negative reference substance DNA into corresponding reaction holes respectively, and performing PCR amplification on the reaction holes.
And (3) amplification procedure:
95 ℃ for 3min (1 cycle);
95 ℃ for 15sec, 58 ℃ for 30sec, 72 ℃ for 30 sec; (35 cycles of the above-mentioned treatment were repeated),
5min at 72 ℃ (1 cycle),
4℃(∞)。
and (3) performing amplification by using a conventional PCR instrument.
(3) Chromatography assay
After the PCR reaction is finished, the following operations are carried out in sequence:
a. taking a substance to be detected (PCR amplification product) for detection, and diluting the amplification product by 10 times by using a sample diluent for later use, wherein the dilution times can be adjusted according to the concentration of a target substance;
b. the test strip is taken out and placed on a horizontal desktop (used as soon as possible), and the NC membrane is not touched;
c. and (3) sucking 80 mu L of sample to be detected by a pipette or a dropper, slowly and dropwise adding the sample to be detected on the sample adding hole of the test strip, and judging and reading the result after 2-10 minutes.
(4) Performance of the kit
As shown in table 4, the results of the positive samples of 13 national references were all positive, and the positive compliance rate was 100%.
TABLE 4 GBS national Positive reference
Figure BDA0002976963930000081
Figure BDA0002976963930000091
As shown in Table 5, the test results of 10 pathogenic bacteria of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Streptococcus pyogenes, Lactobacillus acidophilus, Lactobacillus reuteri, Staphylococcus epidermidis, Escherichia coli DH5 alpha and Candida albicans in the national reference samples were negative, and the coincidence rate was 100%.
TABLE 5 GBS national negative reference
Numbering Pathogens Concentration (CFU/mL) The result of the detection
N1 Streptococcus pneumoniae 1×1010 Negative of
N2 Streptococcus pyogenes 1×1010 Negative of
N3 Streptococcus thermophilus 1×1010 Negative of
N4 Streptococcus mutans 1×1010 Negative of
N5 Streptococcus pyogenes 1×1010 Negative of
N6 Lactobacillus acidophilus 1×1010 Negative of
N7 Lactobacillus reuteri 1×1010 Negative of
N8 Staphylococcus epidermidis 1×1010 Negative of
N9 Large intestine ehichBacterium DH5 alpha 1×1010 Negative of
N10 Candida albicans 1×1010 Negative of
Repeatability: detecting dilution to 1X 107The detection was repeated 10 times for each of GBS1 and GBS5 at CFU/mL, and the detection results were all positive.
Minimum detection limit: the lowest detection limit of the kit is 1 multiplied by 104CFU/mL。
The kit pair is diluted to be not higher than 1 × 104The detection results of GBS1, GBS2 and GBS5 at CFU/mL are all positive.
Example 3
50 clinical samples were tested using the kit described in example 1.
(1) Sample processing
50 vaginal swabs are used for collecting vaginal specimens of 50 pregnant women which are diagnosed in Beijing Kogyang Hospital department in 4 months in 2020 and are born and examined in 35-37 weeks.
The sample collection main points are as follows: the vagina specimen is collected 24 hours before sexual intercourse, bath, vaginal examination, vaginal lavage, local medicine and the like, so that the examination result is not influenced. Generally, saline-soaked cotton swab corner of the eye is used for collecting materials at deep vagina, vaginal fornix, cervical orifice and the like, and is prepared into physiological saline solution of 1mL of vaginal secretion for later use.
A200. mu.L sample was taken, 100. mu.L sample releasing agent was added, and the mixture was allowed to stand on ice for 10min and centrifuged at 10,000g for 2min by a centrifuge. 100 μ L of supernatant was taken for use (the remaining supernatant was stored at-80 ℃ for a long period).
(2) PCR amplification
PCR amplification reaction solutions (20. mu.L per reaction) were prepared as shown in Table 6.
TABLE 6 PCR amplification reaction solution Components
Components 60 reaction volumes
GBS detection liquid 450μL
Amplification reaction MIX 750μL
Total volume 1200uL
The prepared PCR amplification reaction solution was dispensed into each reaction well of 20. mu.L. Adding 5 mul of processed sample DNA, positive reference substance DNA and negative reference substance DNA into corresponding reaction holes respectively, and performing PCR amplification on the reaction holes. And (3) amplification procedure:
95 ℃ for 3min (1 cycle);
95 ℃ 15sec, 58 ℃ 30sec, 72 ℃ 30sec, (35 cycles);
5min at 72 ℃ (1 cycle),
4 ℃ (∞). Amplification was performed using a conventional PCR instrument.
(3) After the PCR reaction is finished, the following operations are carried out in sequence:
a. taking a substance to be detected (PCR product) for detection, diluting a sample with ultrapure water, and determining the dilution times according to the concentration of a target substance;
b. the test strip is taken out and placed on a horizontal desktop (used as soon as possible), and the NC membrane is not touched;
c. and (3) sucking 80 mu L of sample to be detected by a pipette or a dropper, slowly and dropwise adding the sample to be detected on a sample adding hole of the test strip, and judging the result according to the table 2 after 2-10 minutes.
Meanwhile, 50 samples were subjected to comparative detection using a Group B Streptococcus (GBS) nucleic acid detection kit (national mechanical standard 20163402238) purchased from borchenne (beijing) science and technology ltd, and the results were determined strictly according to the instructions.
As shown in Table 7, the final detection results of the two kits showed that 6 of the positive, 41 of the negative, and 3 of the failed detections (probably because no sample was collected), which matched 100% with the detection results of the approved quantitative fluorescence PCR kit.
TABLE 750 clinical samples GBS nucleic acid colloidal gold chromatography and fluorescent quantitative PCR detection results
Figure BDA0002976963930000111
Figure BDA0002976963930000121
As shown in Table 8, 3 samples were re-sampled for the chromatography detection without reference gene, and the detection results were 1 positive and the other 2 negative, thus it can be seen that if no reference gene was used for quality control, a false negative result may be obtained.
TABLE 83 re-sampling test results of amplified samples without reference gene
Figure BDA0002976963930000122
Therefore, the kit can effectively control the whole experimental process of sample collection, nucleic acid extraction and PCR amplification by adding the reference gene, and effectively prevent false negative results.
In summary, with the aid of the above technical solutions, the novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit of the present invention amplifies specific fragments under the action of Taq DNA polymerase after extracting nucleic acids from a collected cell sample, and then realizes group B streptococcus nucleic acid detection by colloidal gold immunochromatography, and in addition, internal reference gene RNAse P is used for tracking quality control in the processes of sample collection, nucleic acid extraction and PCR amplification, thereby ensuring that all experimental processes are in a controllable range. The detection of the GBS nucleic acid amplification product by the kit does not depend on an expensive fluorescent quantitative PCR instrument, the whole operation process is simple, quick, stable and controllable, and in addition, the requirement on the quality of an operator is lower.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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Claims (9)

1. A novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit is characterized by comprising an amplification reaction MIX, a GBS detection solution, a positive control substance, a negative control substance and a sample releasing agent;
the GBS detection solution comprises a reagent against group B streptococcusCAMPSpecific marker primer of gene, specific marker primer of internal reference gene RNAse P and RNase Free ddH2O；
The targeting group B streptococcusCAMPThe specific marker primer sequence of the gene is as follows:
forward primer sequence: 5 '-Bio-CTCTAGTGGCTGGTGCATTGTT-3', as shown in SEQ ID NO.1,
reverse primer sequence: 5 '-TAMARA-GAGTTGTCACTTGATCAGCATGT-3' as shown in SEQ ID NO. 2;
the specific marker primer sequence of the internal reference gene RNAse P is as follows:
forward primer sequence: 5 '-Bio-AGATTTGGACCTGCGAGCG-3', as shown in SEQ ID NO.3,
reverse primer sequence: 5 '-DIG-GAGCGGCTGTCTCCACAAGT-3' as shown in SEQ ID NO. 4.
2. The kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography according to claim 1, wherein the amplification reaction MIX comprises Taq DNA polymerase, 4 dNTPs and ions required by PCR amplification reaction solution.
3. The kit for detecting group B streptococcus nucleic acid PCR-colloidal gold immunochromatography according to claim 1, wherein the positive control is an inactivated group B streptococcus strain.
4. The kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography according to claim 1, whereinThe negative control is RNase Free ddH2O。
5. The kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography according to claim 1, wherein the sample-releasing agent is a cell lysate.
6. The kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography according to claim 1, wherein the GBS detection solution is specific to group B streptococcusCAMPThe concentration of the specific marker primer of the gene is 50-400 nM.
7. The kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography according to claim 1, wherein the GBS detection solution is specific to group B streptococcusCAMPThe concentration of the gene specific marker primer was 200 nM.
8. The novel kit for detecting group B streptococcus nucleic acid by PCR-colloidal gold immunochromatography according to claim 1, wherein the concentration of the specific labeled primer of the internal reference gene RNAse P in the GBS detection solution is 50-400 nM.
9. The novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay kit according to claim 1, wherein the concentration of a specific labeled primer of an internal reference gene RNAse P in the GBS detection solution is 400 nM.
CN202110276766.2A 2021-03-15 2021-03-15 Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatographic assay detection kit Pending CN113025733A (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN110079621A (en) * 2019-04-29 2019-08-02 湖南圣湘生物科技有限公司 Oligonucleotide combinatorial, method and kit for mycobacteria strain identification
CN110734990A (en) * 2019-11-08 2020-01-31 南京农业大学 Detection reagent, kit and application thereof

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Publication number Priority date Publication date Assignee Title
CN110079621A (en) * 2019-04-29 2019-08-02 湖南圣湘生物科技有限公司 Oligonucleotide combinatorial, method and kit for mycobacteria strain identification
CN110734990A (en) * 2019-11-08 2020-01-31 南京农业大学 Detection reagent, kit and application thereof

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