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CN118109620A - PCR-fluorescent probe method kit for optimizing group B streptococcus nucleic acid extraction and amplification detection - Google Patents

PCR-fluorescent probe method kit for optimizing group B streptococcus nucleic acid extraction and amplification detection Download PDF

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CN118109620A
CN118109620A CN202410366782.4A CN202410366782A CN118109620A CN 118109620 A CN118109620 A CN 118109620A CN 202410366782 A CN202410366782 A CN 202410366782A CN 118109620 A CN118109620 A CN 118109620A
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pcr
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马伟民
马欣宜
魏其林
许元峰
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Shenzhen Blot Biotech Co ltd
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    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

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Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a PCR-fluorescent probe method kit for optimizing group B streptococcus nucleic acid extraction and amplification detection. The invention provides a primer probe combination for targeting GBS, a qPCR reaction reagent, a detection kit for GBS and a detection method, wherein the accuracy, the sensitivity and the specificity of detection are improved by designing a double-target and endogenous internal standard multiple amplification system and matching with a proper PCR reaction reagent and an extraction reagent. And then, through the combination screening of a plurality of pairs of primer probes and the optimization of an amplification system, the detection kit can detect bacterial liquid of 100 CFU/ml.

Description

PCR-fluorescent probe method kit for optimizing group B streptococcus nucleic acid extraction and amplification detection
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a PCR-fluorescent probe method kit for optimizing group B streptococcus nucleic acid extraction and amplification detection.
Background
Streptococcus agalactiae (Streptococcus agalactiae) belongs to group B in the Lanceolatae antigen classification, which is currently only found in this bacterium, and is therefore also commonly referred to as group B Streptococcus (Group B Streptococcus, GBS). GBS is a beta-hemolytic gram-positive coccus, paired or arranged in short chain, and can be classified into Ia, ib, ic, II, III, IV, V, VI, VII, VIII and IX according to the capsular polysaccharide of the bacteria.
Group B streptococcus is a gram positive bacterium which normally colonizes the vagina and rectum of human beings and belongs to conditional pathogenic bacteria. In the 70 s of the 20 th century, GBS has been identified as an important pathogen for perinatal maternal and infant infections, and plays a non-negligible role in perinatal medicine. GBS mainly infects infants, pregnant women, and postpartum women, causing invasive disease. For special people such as pregnant and lying-in women, the genital tract infection GBS can cause ascending infection of the genital tract of the pregnant women, and cause uterine cavity infection, amniotic infection, endometritis, pneumonia and even systemic serious infection, such as septicemia, bacteremia and the like; for the fetus in the abdomen, bad pregnancy outcomes such as premature birth, dead fetus, premature rupture of fetal membranes, abortion and the like can be caused, and the fetus is delivered to the neonate by the pregnant woman when the fetus is delivered through the birth canal, so that the neonate GBS disease is caused.
The american society for microbiology ASM, in 2021, 7.23, has recently issued a recent guideline for group B streptococcus detection and identification, which proposes vaginal-anal swab sampling and GBS screening of all pregnant women at 36-37 +7 weeks, with antibiotic preventative treatment of patients with positive results. The department of obstetrics and gynecology of the China medical society, department of obstetrics and gynecology, and group of science for preventing group B streptococcosis in perinatal period (China) expert consensus, recommended to conduct vaginal-rectal GBS screening on all pregnant women in 35-37 weeks. Therefore, a method for rapidly and accurately detecting streptococcus agalactiae is established, and has very important clinical application value.
The GBS detection products on the market at present are mainly based on the following 3 detection technologies: culture chromogenic method, immunoreaction method and fluorescence PCR method. The culture chromogenic method is gold standard, but has high nutrition requirement on GBS, is easy to generate false negative result under the condition of mixed bacteria pollution, has long test time and needs up to 2 days. The immune reaction method has the advantages of quick test, no special instrument requirement and low sensitivity, and the fatal disadvantage. Compared with a culture chromogenic method and an immune reaction method, the fluorescent PCR method needs to be used for nucleic acid extraction or amplification culture and needs to be matched with a real-time fluorescent quantitative PCR instrument, and has the advantages of short detection time, strong specificity and high sensitivity. The real-time quantitative PCR detection platform has become the simplest and most reliable bacterial detection method. However, the existing reagent for detecting GBS based on the fluorescent quantitative PCR method still has a plurality of defects:
First, the limit of detection (i.e., the minimum detection concentration) of group B streptococcus on the market is mostly centered at 1000CFU/ml, and for some patients with weak positive infections, false negative detection results may exist, and the optimal antibiotic intervention time is missed, resulting in invasive infections of pregnant and parturients and newborns.
Secondly, most of detection targets of commercial group B streptococcus nucleic acid detection kits (PCR-fluorescent probe method) which have obtained national medical instrument registration evidence are CFB genes, namely single targets, and there is a possibility that gene mutation exists and cannot be detected, so that detection is missed and diagnosis of doctors is interfered.
In addition, in the group B streptococcus nucleic acid detection kit (PCR-fluorescent probe method) with the national medical instrument registration certificate, several kits with better sales are all in a state of independently storing the hot start Taq enzyme and are not mixed with a reaction buffer system mix, and an operator needs to remix the reaction solution according to the proportion, so that the risk of misoperation or the risk of mismatching of the volumes of all components can be increased.
Finally, a common complication in PCR amplification laboratories is contamination of the next batch of reagents with amplified products, leading to false positive results. Most of the current reagents cannot avoid the problem.
It can be seen that a further improvement in the PCR-fluorescent probe kit for the extraction and detection of group B streptococcal nucleic acids is necessary.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a PCR-fluorescent probe method kit for optimizing the extraction and amplification detection of group B streptococcus nucleic acid.
The invention provides a primer probe combination, which comprises a primer and a probe for targeting CFB genes and SIP genes, wherein:
The upstream primer of the targeting CFB gene is GGTTAATGAGGCTATTACTAGTGTTGA '-3' (shown as SEQ ID NO: 1)
The downstream primer of the targeting CFB gene is ATCACATCTGTTAAGGCTTCTACACG '-3' (shown as SEQ ID NO: 2);
The probe of the targeting CFB gene is TGCGTGCCAACCCTGAGACAGTTTATG '-3' (shown as SEQ ID NO: 3);
the upstream primer of the target SIP gene is CACCAGCTCCTGTGAAGTCG '-3' (shown as SEQ ID NO: 4);
The downstream primer of the target SIP gene is AGCTGGTGTTTCAGCGGCAAC '-3' (shown as SEQ ID NO: 5);
the probe of the target SIP gene is GTCAGTCAGTCAGTCAACAACAGTATCAC '-3' (shown as SEQ ID NO: 6).
The primer probe combination also comprises a primer and a probe for targeting the reference gene, the selection of the reference gene is not limited, and the primer probe combination belongs to the cell stably expressed gene and plays the same role in monitoring, and belongs to the technical scheme. In some embodiments, the reference gene is selected from at least one of 18s, b-action, GAPDH, cyclopylin, HPRT, GUS, or RNaseP.
Furthermore, the primer probe combination provided by the invention further comprises a primer and a probe for targeting the RNaseP gene.
The upstream primer of the targeting RNaseP gene is TGGCGGTGTTTGCAGATTT (shown as SEQ ID NO: 7) from 5 '-3';
the downstream primer of the targeting RNaseP gene is GCGGCTGTCTCCACAAGTC (shown as SEQ ID NO: 8) from 5 '-3';
The probe of the targeting RNaseP gene is GTTCTGACCTGAAGGCTCTGCGC '-3' (shown as SEQ ID NO: 9).
In the primer probe combination, the 5 'end of the probe carries a fluorescent group, and the 3' end carries a quenching group.
The fluorescent group is selected from AMCA、Atto 425、FAM、Alexa Fluor 488、Pacific Blue、Cy3、TET、JOE、HEX、Quasar 570、TAMRA、ROX、Texas red、Atto 590、Cy5、Quasar 670 or Cy5.5; the quenching group is selected from any one of Dabcyl, eclipse, MGB, BHQ, BHQ2 or BHQ 3.
In the invention, the probe of the target gene carries FAM fluorescent groups and BHQ1 quenching groups. The internal reference gene RNaseP probe carries a VIC fluorescent group and a BHQ1 quenching group.
The invention designs specific primers and probes in consideration of various factors (including traditional and new variant genotypes and serotypes) and the like, designs a double-target and endogenous internal standard multiple amplification system aiming at the CFB gene and SIP gene double targets and the human endogenous reference gene RNaseP gene, and improves the detection accuracy. And then, through the combination screening of a plurality of pairs of primer probes and the optimization of an amplification system, the detection kit can detect bacterial liquid of 100 CFU/ml. Compared with the traditional kit for detecting only a single target, the kit has the advantages of high detection sensitivity, good specificity, wide inclusion (various serotypes Ia, ib, ic, II, III, IV, V, VI, VII, VIII and IX of group B streptococcus can be detected), strong repeatability and quick detection result.
Further, the invention also provides a qPCR reaction reagent of the group B streptococcus, which comprises the primer probe combination, a PCR reaction liquid, a PCR enhancer and a buffer system.
In the qPCR reagent of the present invention, the primer probe combination, the PCR reaction solution, the PCR enhancer and the buffer system are independent of each other, or may be mixed with each other, which is not limited in the present invention. In the embodiment of the invention, the concentration of each component in the qPCR reaction reagent is the working concentration, and the reagent can be stored at the working concentration or 2-100 times of the working concentration in the storage process. For example, 2×,5×,10×, 20×,50×, 100×.
The PCR reaction liquid comprises the following components: UDG enzyme, hot start Taq enzyme, taq enzyme antibody protectant and deoxynucleoside triphosphates.
In the PCR reaction liquid, the hot-start taq enzyme is used and the hot-start taq enzyme antibody protective agent is added, so that the problem that the PCR amplification mix and the taq enzyme are stored separately in the traditional kit is solved.
The PCR reaction liquid of the invention is added with a UDG enzyme anti-pollution system, thus reducing false positive caused by pollution of amplified products. And the PCR fluorescence detection is performed in a fully closed mode, a tube cover is not required to be opened after the extracted sample is added, and only 2 hours are required from sample treatment to fluorescence detection completion. Experiments show that in the reaction at 50 ℃ and 2min before the PCR reaction, UDG enzyme can hydrolyze uracil base and N-glycosidic bond of sugar phosphate skeleton of PCR product containing uracil in the PCR reaction liquid to release free uracil. Then heat treatment is carried out at 95 ℃ for 10min, and the phosphate skeleton is further hydrolyzed while the UDG enzyme is inactivated, so that the pollution of PCR products containing uracil is eliminated, and other enzymes are not interfered to play roles.
In some embodiments, wherein the concentration of UDG enzyme is 0.01-0.1U/. Mu.l, the concentration of hot start Taq enzyme is 0.01-0.1U/. Mu.l, the concentration of Taq enzyme antibody protectant is 0.01-0.2U/. Mu.l, the concentration of deoxynucleoside triphosphates is 100-900. Mu.M, dATP: dUTP: dGTP: dCTP=1:1:1:1. Preferably, the concentration of UDG enzyme is 0.02U/. Mu.l, the concentration of hot start Taq enzyme is 0.03U/. Mu.l, the concentration of Taq enzyme antibody protecting agent is 0.03U/. Mu.l, and the concentration of deoxynucleoside triphosphates is 200. Mu.M.
The PCR enhancer of the invention comprises: betaine or a hydrate thereof, glycerol and tetramethylammonium chloride.
The qPCR reagent contains a PCR enhancer, and comprises betaine, glycerol and tetramethyl ammonium chloride (TMAC). Among them, betaine or hydrobetaine can enhance PCR amplification of GC-rich DNA sequences by reducing secondary structure formation caused by GC-rich regions. Glycerol can reduce the secondary structure of DNA double strand and reduce non-specific amplification products. Tetramethyl ammonium chloride (TMAC) reduces DNA mismatch during the amplification reaction to improve specificity. Several components are mutually matched to form a water-in-oil structure in a PCR reaction system. Before the amplification reaction, the PCR enhancer can form a non-interactive compartment in the molecule, and simultaneously forms a relatively isolated space with the outside environment of the tube, so that the component concentration change or property change caused by liquid evaporation and the like is reduced; in the amplification reaction, the PCR enhancer and the amplification reagent are fully mixed, so that enzymatic reactions such as DNA polymerase and the like are promoted, and the amplification efficiency is improved. Compared with other reinforcing agents, the combination of betaine, glycerol and tetramethyl ammonium chloride can more effectively lead the Ct value of the amplification reaction to be relatively advanced, the amplification curve is closer to a standard S-shaped curve, and the detection effect is greatly optimized.
In some embodiments, the working concentrations of the components include: the concentration of betaine or hydrate thereof is 1-2 mol/L, the volume fraction of glycerol is 20-30%, and the concentration of tetramethyl ammonium chloride is 25-35 mM. In some embodiments, the PCR enhancer consists of 1.4M betaine or betaine monohydrate, 25 vol.% glycerol, 30mM tetramethyl ammonium chloride, and water;
In the qPCR reaction reagent of the invention, the buffer system comprises: tris-HCl, magnesium chloride, potassium chloride, sodium chloride, triton, and formamide.
The buffer system has reasonable component arrangement and can well match with enzyme, primer, probe in the reaction reagent, thereby realizing the mixing of the components such as Taq enzyme, reaction buffer system mix and the like in the same tube reaction liquid, and the stable preservation and the simplified operation process. The composition structure or the composition concentration of the buffer system is adjusted, which results in a decrease in storage stability and influences the detection result.
In some embodiments, the working concentrations of the components include: the concentration of Tris-HCl is 40-60 mmol/l and the pH value is 8.0. The concentration of magnesium chloride is 15-25 mmol/l, the concentration of potassium chloride is 15-25 mmol/l, the concentration of sodium chloride is 100-200 mmol/l, the concentration of triton is 0.1-0.5 vol%, and the concentration of formamide is 8-12 vol%.
In some embodiments, the working concentrations of the components include: the buffer system consists of 50mmol/l Tris-HCl, 20mmol/l magnesium chloride, 20mmol/l potassium chloride, 150mmol/l sodium chloride, 0.2vol% triton, 10vol% formamide and water.
In the present invention, the working concentration of each component in the detection reagent is the concentration after premixing, and during the preservation process, the PCR reaction solution, the PCR enhancer and the buffer system are in a mixed state, and the concentration of each component after mixing is the working concentration or is 2-100 times of the working concentration, for example, the concentration after mixing is 2 times, 10 times, 20 times, 50 times or 100 times of the working concentration.
The invention prepares the PCR amplification mix buffer system, taq enzyme, uracil-N glycosylase (UDG) and other reagents into one tube in advance, improves the experimental efficiency, saves the time of repeated liquid preparation of a user and reduces the use error. The primer probe adopted in the reagent targets two different target genes, and the accuracy test proves that the primer probe has the detection effect on different types of streptococcus B, including Ia, ib, ic, II, III, IV, V, VI, VII, VIII and/or IX and the like, has strong pertinence and high specificity, and is not easy to generate cross reaction with strains of different species of the same genus.
Still further, the present invention provides a group B streptococcus detection kit comprising a primer probe combination as described above, or comprising a qPCR reaction reagent as described above.
In some embodiments, the detection kit of the present invention further comprises a nucleic acid extraction reagent; the nucleic acid extraction reagent comprises: pretreatment liquid, lysozyme, lysate, proteinase K, magnetic beads and rinsing liquid;
In some embodiments, the lysate comprises: surfactant, polyethylene glycol 4-tert-octylphenyl ester and guanidine hydrochloride.
The nucleic acid extraction reagent contained in the kit uses two surfactants, namely surfactant and polyethylene glycol 4-tert-octylphenyl ester (Triton X-45), as components of a lysate, and is used for lysing cells, fully separating nucleic acid from other substances in the cells such as protein, lipid and the like, and increasing the yield of the nucleic acid after the lysis, thereby improving the detection capability of the kit on low-concentration bacterial liquid or samples, and reducing the concentration of the detectable minimum bacterial liquid to 100CFU/ml. In order to better lyse the sample and ensure the integrity of nucleic acid, thereby improving the detection effect, the composition and the concentration of the lysate are screened. Preferably, the lysate consists of 0.8-1.2 wt% of surfactant, 1-2 wt% of polyethylene glycol 4-tert-octyl phenyl ester, 4-6 mol/L guanidine hydrochloride and water. More preferably, the lysate consists of 0.8wt% of surfactant, 1.5wt% of polyethylene glycol 4-t-octylphenyl ester, 4mol/L guanidine hydrochloride, and water.
Aiming at the defects of insufficient cracking, complicated operation, inapplicability to a large number of samples and the like of an anal swab, a vaginal swab and the like of an alkali cracking method or an improved nucleic acid extraction method based on the alkali cracking method, the nucleic acid extraction reagent in the kit provided by the invention adopts a magnetic bead method to separate nucleic acid, and the modified magnetic bead is specifically combined with the nucleic acid and separated under the action of an external magnetic field. The kit can be matched with an automatic nucleic acid extraction instrument for separating nucleic acid by a magnetic bead method, and further has the characteristics of high flux, high quality and high stability. In particular to gram-positive bacteria, the invention avoids the interference of mucus and other impurities by improving the formula of the lysate and combining with the nucleic acid extraction of a magnetic bead method, so that the gram-positive bacteria are easier to break the wall, the nucleic acid is fully exposed, and the concentration of the final product is improved.
In the invention, the kit also comprises a pretreatment liquid, wherein the pretreatment liquid comprises 100mM Tris, 5mM EDTA, 1-1.5 vol% TritonX-100 and water.
In the invention, the kit also comprises a rinsing liquid, wherein the rinsing liquid comprises the following components: 100mM Tris, 500mM Na 2 -EDTA, 1 to 1.5vol% TritonX-100, 4 to 8mM MOPS, 3 to 6M guanidine hydrochloride and water.
In the invention, the kit also comprises an eluent, wherein the eluent is DEPC water.
The kit also comprises positive control and negative control;
The positive control is a mixture of CFB plasmid, SIP plasmid and RNaseP plasmid; wherein the copy number of each plasmid is (1.00-5.00) E5 copies/ml.
The negative control is a human cell line culture. The cell concentration was (1.00-5.00) E5 cells/ml.
The invention also provides a method for detecting GBS bacteria,
Comprising detecting a sample with a primer probe combination as described above;
Or comprises detecting the sample with a qPCR reaction reagent as described previously;
or involve the detection of a sample with a detection kit as described previously.
The detection method of the present invention includes diagnostic purposes, but may also be non-diagnostic purposes, and the present invention is not limited thereto. Detection for non-diagnostic purposes includes detection of laboratory samples, environmental samples, medical devices, and the like. The diagnostic purpose is detected, and the sample is a vaginal swab or an anal swab.
The PCR reaction system in the method for detecting GBS bacteria comprises the following steps:
The PCR reaction program in the method for detecting GBS bacteria comprises the following steps:
The method further comprises the step of extracting sample DNA before the PCR reaction.
The DNA extraction includes: and cleaning and resuspension the sample with a pretreatment liquid, carrying out enzymolysis by lysozyme, then treating with a lysate and proteinase K, and eluting after magnetic bead separation and rinsing to obtain DNA.
The invention provides a primer probe combination for targeting GBS, a qPCR reaction reagent, a detection kit and a detection method for GBS. The scheme of the invention has at least the following advantages:
Firstly, the invention designs specific primers and probes in consideration of various factors (including traditional and new variant genotypes and serotypes) and the like, designs a double-target and endogenous internal standard multiple amplification system, and improves detection accuracy. And then, through the combination screening of a plurality of pairs of primer probes and the optimization of an amplification system, the detection kit can detect bacterial liquid of 100 CFU/ml.
Secondly, the invention adopts a CFB gene and SIP gene double-target and human endogenous reference gene RNaseP gene multiple amplification system, and compared with the traditional kit for detecting only a single target, the kit has the advantages of high detection sensitivity, good specificity, wide inclusion (multiple serotypes Ia, ib, ic, II, III, IV, V, VI, VII, VIII and IX of group B streptococcus can be detected), strong repeatability and rapid detection result. The human endogenous reference gene RNaseP gene is added with a sample acquisition monitoring system to control false negative results caused by experimental operation and measurement equipment problems, so that the detection results are more reliable and stable.
Thirdly, the hot-start taq enzyme is used in the detection reaction system, and the hot-start taq enzyme antibody protective agent is added, so that the problem that the PCR amplification mix and the taq enzyme are stored separately in the traditional kit is solved. The kit is simple and easy to use and convenient to operate.
Fourth, the detection kit and the addition of the UDG enzyme anti-pollution system reduce false positive caused by pollution of amplified products. And the PCR fluorescence detection is performed in a fully closed mode, a tube cover is not required to be opened after the extracted sample is added, and only 2 hours are required from sample treatment to fluorescence detection completion. Meanwhile, the PCR enhancer is added into the detection kit, and the detection kit comprises betaine, glycerol and tetramethyl ammonium chloride (TMAC) to form a water-in-oil structure, so that liquid evaporation is reduced, taq enzymatic reaction is fully promoted, and further amplification efficiency is improved.
Fifth, the nucleic acid extraction reagent contained in the kit uses two surfactants, namely surfactant and polyethylene glycol 4-tert-octylphenyl ester (Triton X-45), as components of a lysate, and is used for lysing cells, fully separating nucleic acid from other substances in the cells such as protein, lipid and the like, and increasing the yield of the nucleic acid after the lysis, thereby improving the detection capability of the kit on low-concentration bacterial liquid or samples, and reducing the concentration of the detectable minimum bacterial liquid to 100CFU/ml.
Drawings
FIG. 1 is a clinical sample test chart;
FIG. 2 positive reference plot;
FIG. 3 is a negative reference plot;
FIG. 4 is a specificity diagram;
FIG. 5 sensitivity chart;
FIG. 6 is a comparative primer probe combination diagram of the CFB gene;
FIG. 7 is a comparison of primer probe combinations for SIP genes;
FIG. 8 primer probe combination comparison of RNaseP gene;
FIG. 9 is a comparison of different PCR enhancer protocols versus national reference GBS1 of 1X 10 6 CFU/mL;
FIG. 10 is a comparison of different PCR enhancer protocols versus national reference GBS5 of 1X 10 6 CFU/mL;
FIG. 11 is a comparison of different PCR enhancer protocols versus national reference GBS7 of 1X 10 6 CFU/mL.
Detailed Description
The invention provides a PCR-fluorescent probe method kit for optimizing the extraction and amplification detection of group B streptococcus nucleic acid, and the technical parameters can be properly improved by the person skilled in the art by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
Example 1 group B Streptococcus nucleic acid detection kit (PCR-fluorescent Probe method).
The group B streptococcus nucleic acid detection kit (PCR-fluorescent probe method) comprises PCR amplification reaction liquid, positive control and negative control. The PCR reaction solution comprises a primer, a probe, UDG enzyme, hot start Taq enzyme, taq enzyme antibody protective agent, deoxynucleoside triphosphate (containing dNUTP), PCR enhancer (betaine, glycerol, tetramethyl ammonium chloride (TMAC)), mg 2+ buffer system and the like.
Table 1 composition of kit
The primer probe sequences of the CFB target gene and the SIP target gene and the internal reference RNaseP internal reference gene are as follows:
TABLE 2 primer probe sequences for kits
Name of the name Sequence (5 '-3') Final concentration
Primer CFB-FP GGTTAATGAGGCTATTACTAGTGTTGA 250nM
Primer CFB-RP ATCACATCTGTTAAGGCTTCTACACG 250nM
Probe CFB-P FAM-TGCGTGCCAACCCTGAGACAGTTTATG-BHQ1 200nM
Primer SIP-FP CACCAGCTCCTGTGAAGTCG 250nM
Primer SIP-RP AGCTGGTGTTTCAGCGGCAAC 250nM
Probe SIP-P FAM-GTCAGTCAGTCAGTCAACAACAGTATCAC-BHQ1 200nM
Primer RNaseP-FP TGGCGGTGTTTGCAGATTT 200nM
Primer RNaseP-RP GCGGCTGTCTCCACAAGTC 200nM
Probe RNaseP-P VIC-GTTCTGACCTGAAGGCTCTGCGC-BHQ1 150nM
Example 2 group B Streptococcus nucleic acid extraction assay
The present embodiment provides the operation steps of the nucleic acid extraction reagent and the effect on the detection limit:
1. Sample pretreatment:
Adding 1-3ml physiological saline into the dry swab sample, and uniformly mixing;
The sample containing preservation solution (containing guanidine hydrochloride, tris, EDTA, SDS, phenol red) is filled to at least 1ml;
Taking a bacterial sample and a bacterial culture solution;
The above samples were centrifuged at 1ml at 12,000rpm (13,400Xg) for 1mm and the supernatant was discarded. 110. Mu.L of pretreatment solution and 70. Mu.L of lysozyme solution were added and treated at 37℃for 30 minutes or more.
2. 600. Mu.L of lysate and 20. Mu.L of proteinase K were added and shaken until the precipitate was thoroughly suspended and left at 65℃for more than 15min until the liquid became clear. Wherein the lysate contains 0.8% of surfactant (surfactin), 1.5% of polyethylene glycol 4-tert-octylphenyl ester (Triton X-45), 4mol/L guanidine hydrochloride and water, and has pH of 7.5+ -0.5.
3. Adding 20 mu L of magnetic bead solution (the magnetic beads are fully and uniformly mixed before use) into a centrifuge tube, and vibrating and uniformly mixing for 5min; the centrifuge tube was placed on a magnetic rack and allowed to stand for 1min, after which the beads were fully adsorbed, the liquid was carefully aspirated.
4. Adding 800 μl of rinsing solution I (before use, checking whether absolute ethanol is added) and shaking for mixing for 2min; the centrifuge tube was placed on a magnetic rack and allowed to stand for 1min, after which the beads were fully adsorbed, the liquid was carefully aspirated.
5. Adding 800 μl of rinsing solution II (before use, checking whether absolute ethanol is added) and shaking for 2min; the centrifuge tube was placed on a magnetic rack and allowed to stand for 1min, after which the beads were fully adsorbed, the liquid was carefully aspirated.
6. Adding 500 μl of rinsing solution II (before use, checking whether absolute ethanol is added), shaking, and mixing for 2min; the centrifuge tube was placed on a magnetic rack and allowed to stand for 1min, after which the beads were fully adsorbed, the liquid was carefully aspirated.
7. Placing the centrifuge tube on a magnetic rack, and airing for 5-10 min at room temperature.
8. Taking down the centrifuge tube from the magnetic rack, adding 50-100 mu L of eluent, shaking and mixing uniformly, placing the mixture at 65 ℃ for incubation for 5min, and reversing and mixing uniformly for 3 times each time for 3-5 times.
9. The centrifuge tube was placed on a magnetic rack for 2min, after complete adsorption of the beads, the DNA solution was carefully transferred to a fresh centrifuge tube and stored under appropriate conditions.
The extracted GBS nucleic acid is amplified according to the instructions by using the kit:
Amplification reaction system:
table 3 amplification reaction System of kit
Amplification reaction procedure:
Table 4 kit amplification reaction procedure
CT values under different bacteria concentrations can be obtained by detection, and the lowest concentration which can be detected by 95% is taken as the detection limit of the kit, and the results are shown in the following table. The analysis is easy to obtain, when the bacterial concentration of GBS is 100CFU/ml, the detection rate can reach 95%, and when the bacterial concentration of GBS is lower than 100CFU/ml, the detection rate is lower than 95%, so that the detection limit of GBS can be reduced to 100CFU/ml by extracting and amplifying the nucleic acid of GBS through the kit.
TABLE 5 GBS detection rate at different bacterial concentrations
Example 3 group B Streptococcus clinical sample detection
This example provides the group B streptococcus nucleic acid detection kit (PCR-fluorescent probe method) of example 1 above for use in the detection of group B streptococcus in a mixed sampling swab of clinical specimens genital tract secretions and rectal secretions, comprising the steps of:
1. Sample processing
To the mixed sampling swab tube, 1ml of physiological saline was added, and the mixture was thoroughly mixed by shaking, and it was recommended to take 300ul of the sample mixture for nucleic acid extraction. Nucleic acid extraction reagents are employed. Note that the negative control was extracted simultaneously and 5 μl of the supernatant was collected for detection.
2. Reagent preparation
And taking out GBS PCR reaction liquid from the kit, oscillating and mixing after melting at room temperature, and carrying out instantaneous centrifugation for a plurality of seconds. The sample size (n) was measured as needed, and the (n+2) tube was dispensed into PCR tubes at 15. Mu.L/tube.
3. Sample addition
And respectively adding 10 mu l of the extracted sample nucleic acid to be detected, GBS positive control nucleic acid and negative control nucleic acid into the corresponding GBS PCR reaction tube, covering a tube cover tightly, and transferring to an amplification detection area after instantaneous centrifugation for a plurality of seconds.
4. Fluorescent PCR amplification
And placing the PCR reaction tube in a sample tank of a fluorescent quantitative PCR instrument, and setting the names of samples to be detected according to the corresponding sequence. PCR amplification parameters were set as follows in tables 6-7.
Table 6 example three-sample kit PCR amplification parameters
TABLE 7 example 3 kit fluorophore parameters
5. After the amplification is finished, judging whether the streptococcus B is infected according to a fluorescence curve. The amplification curve is shown in FIG. 1.
6. Result analysis condition setting
ABI7500: baseline (Baseline) was adjusted according to post-analysis images: start=3, end=15; adjustment Threshold (Threshold): the FAM channel is set to 10000, the VIC channel is set to 5000, and the FAM channel can be adjusted if special conditions are met.
SLAN-96S: baseline (Baseline) was adjusted according to post-analysis images: start=6, end=12; the threshold value is generally selected from default values, and can be specifically adjusted according to actual conditions.
6.1 Quality control
TABLE 8 example 3 kit fluorophore parameters
Note that: the above requirements must be met simultaneously in the same experiment, otherwise the experiment is regarded as ineffective.
6.2 Interpretation of test results
According to the set base line and threshold, the instrument automatically displays the detected Ct value, and the result judgment can be carried out according to the Ct value:
Table 9 example 3 kit results interpretation basis
① The experiment is invalid and the detection needs to be repeated: FAM channel has no typical S-type amplification curve or 38< Ct < 45; the VIC channel has no typical S-type amplification curve or ct= Undet.
② The VIC channel is Ct > 35 or undetected, indicating that the sample contains PCR reaction inhibitors, suggesting that the sample be re-extracted.
③ And a control is set for quality control (positive quality control product and blank control) in each experiment, and when the quality control product result does not accord with the quality standard, the experiment is invalid.
Table 10 clinical sample test results statistics
Example 4 group B Streptococcus nucleic acid detection kit (PCR-fluorescent Probe method) accuracy experiment
In order to detect the specificity of the group B streptococcus detection kit, the kit prepared in the embodiment 1 is adopted to detect the working reference of enterprises, and the coincidence rate of the yin-yang reference is 100%. The kit detects 10 cases of working references of a yin-yang enterprise, and the detection results are shown in the following table and figures 2-3:
TABLE 11 working reference results statistics for yin-yang enterprises
Positive reference Results Negative reference Results
P1 + N1 -
P2 + N2 -
P3 + N3 -
P4 + N4 -
P5 + N5 -
R1 + N6 -
R2 + N7 -
R3 + N8 -
R4 + N9 -
R5 + N10 -
Conclusion: as can be seen from FIGS. 2 and 3, the nucleic acid extraction reagent of example 1 was used to detect 10 samples of female and male working references of an enterprise, and the amplification curve was a standard S-shaped curve, wherein the negative coincidence rate was 100%, the positive coincidence rate was 100%, and the accuracy of the kit was high.
Example 5 group 5 Streptococcus nucleic acid detection kit (PCR-fluorescent Probe method) specificity experiment
Clinically common pathogens and microorganisms were detected with the kit of example 1, and the microorganisms include: streptococcus pneumoniae, streptococcus grass, enterococcus faecium (NG), chlamydia trachomatis, ureaplasma urealyticum, mycoplasma hominis, trichomonas vaginalis, candida albicans, and herpes simplex virus type ii. The results show that the detection result is negative, and the detection result has no cross reaction with all pathogens or microorganisms. The PCR fluorescent probe method for detecting the nucleic acid by the group B streptococcus is shown to have strong specificity.
Example 6 sensitivity test of group 6B Streptococcus nucleic acid detection kit (PCR-fluorescent Probe method)
The limits of detection of the kit were studied with the kit of example 1 on group B streptococcus (including GBSIa for GBS1, GBS II for GBS2, GBS III for GBS5, GBS V for GBS 7) using different serotypes. 5 concentration gradients of 1X 10CFU/mL and 1X 10 2CFU/mL、1×103CFU/mL、1×104CFU/mL、1×105 CFU/mL for each of GBS1, GBS2, GBS5 and GBS7 were selected, and the detection was repeated 3 times after each concentration reference was extracted, and the lowest concentration of 3 repeated complete detections was determined. The kit can detect bacterial liquid with the sensitivity of 100 CFU/ml.
Table 12 sensitivity results statistics
Example 7 group 7 detection of plasmids with Streptococcus nucleic acid detection kit (PCR-fluorescent Probe method)
Using the plasmid samples as templates, amplification was performed under the conditions of the kit of example 1, and the limits of detection of plasmids by the kit were investigated as follows:
1. Plasmid samples: contains CFB gene, SIP gene and human endogenous reference gene RNaseP gene of group B streptococcus, and can be amplified by qPCR under proper conditions through corresponding primers and probes to obtain a real-time fluorescent quantitative PCR curve. CFB genes, SIP genes were diluted to different copy numbers, 500, 250, 100copies/μl, using TE buffer, depending on copy number.
2. Using the above plasmid samples as templates, each concentration was tested for 21 replicates and amplified according to the amplification procedure of example three.
3. Analysis of results: the result with CT value less than or equal to 38 is judged as positive, and the detection rate of plasmid is counted, and the result is shown in the following table.
TABLE 13 detection rate of CFB Gene, SIP Gene, RNaseP Gene at different plasmid concentrations
Example 8 control experiment of primer probe of the kit and detection Rate comparison with certain commercially available kit
Based on CFB gene and SIP gene of group B streptococcus and internal reference gene RNaseP, designing a plurality of pairs of primer probes as shown in table 14, and detecting the national reference GBS strain to obtain the combination with optimal curve morphology and minimum Ct value, as shown in figures 6-8.
Table 14 designed multiple pairs of primer probe combinations
And combining the optimized primer probes to prepare PCR reaction liquid, and performing amplification detection on 220 clinical samples. Meanwhile, the same clinical sample was detected by a commercial group B streptococcus nucleic acid detection kit (PCR-fluorescent probe method). The statistical test results are shown in Table 15. The result shows that compared with the commercial kit, the kit can increase the detection rate of clinical samples by increasing the SIP genes, and avoid missed detection.
TABLE 15 comparison of the detection results of the kit and a commercially available kit
Clinical samples CFB Gene results SIP Gene results The result is interpreted by the kit Interpretation of results by certain commercially available kits
Positive, 40 cases 37 23 40 38
Negative, 180 cases 183 197 180 182
Example 9 comparison of the detection Effect of different lysate fractions on GBS national reference at different concentrations
Lysates of different compositions and concentrations were used as shown in table 16.
TABLE 16 different lysate protocols
Group B streptococci (Ia), group B streptococci (III), group B streptococci (V) in national references were diluted to 100CFU/mL according to the instructions using RNase/DNase free deionized water. Nucleic acid extraction is carried out by the lysate, and then the PCR reaction liquid of the kit is used for amplification. The statistical test results are shown in Table 17. The results show that the lysate of the scheme 3 and the scheme 4 can lead the detection rate of the group B streptococcus (Ia), the group B streptococcus (III) and the group B streptococcus (V) to reach more than 95 percent per mL. Meanwhile, the average value of Ct values can be minimized in the scheme 3.
TABLE 17 detection effect of different lysate protocols on minimum detection limit of national reference
Example 10 comparison of the detection Effect of different PCR enhancer Components on GBS national reference
The nucleic acid extraction reagent of example 1 was used to extract national references GBS1, GBS5 and GBS7 at a concentration of 1X 10 6 CFU/mL. The extracted nucleic acids were amplified using different components and concentrations of PCR enhancers, as in table 18, configured as PCR reaction solutions. The results show that the PCR enhancer formula of the scheme 3 can enable the curve form to be S-shaped, smoother and have fewer broken lines.
TABLE 18 different PCR enhancer protocols
PCR enhancer Scheme 1 Scheme 2 Scheme 3 Scheme 4 Scheme 5
Betaine (betaine) 0M 0.8M 1.4M 1.4M 1.4M
Glycerol 25％ 25％ 25％ 0％ 25％
Tetramethyl ammonium chloride (TMAC) 30mM 30mM 30mM 30mM 0mM
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Primer probe combinations comprising primers and probes targeting CFB genes and SIP genes, wherein:
the upstream primer of the targeting CFB gene is shown as SEQ ID NO. 1;
the downstream primer of the targeting CFB gene is shown as SEQ ID NO. 2;
The probe of the targeting CFB gene is shown as SEQ ID NO. 3;
The upstream primer of the target SIP gene is shown as SEQ ID NO. 4;
The downstream primer of the target SIP gene is shown as SEQ ID NO. 5;
The probe of the target SIP gene is shown as SEQ ID NO. 6.
2. The primer probe combination of claim 1, further comprising a primer and a probe targeting the RNaseP gene,
The upstream primer of the targeting RNaseP gene is shown as SEQ ID NO. 7;
The downstream primer of the targeting RNaseP gene is shown as SEQ ID NO. 8;
The probe of the targeting RNaseP gene is shown as SEQ ID NO. 9.
A qPCR reagent for group b streptococcus comprising the primer probe combination of claim 1 or 2, a PCR reaction solution, a PCR enhancer, and a buffer system;
The PCR reaction liquid comprises the following components: UDG enzyme, hot start Taq enzyme, taq enzyme antibody protectant and deoxynucleoside triphosphates;
The PCR enhancer comprises: betaine or a hydrate thereof, glycerol and tetramethylammonium chloride;
The buffer system comprises: tris-HCl, magnesium chloride, potassium chloride, sodium chloride, triton, and formamide.
4. The detection reagent according to claim 3, wherein the primer probe combination of claim 1 or 2, the PCR reaction solution, the PCR enhancer and the buffer system are premixed, wherein:
The working concentration of each primer and each probe is 100 nM-900 nM respectively;
The working concentration of the components of the PCR reaction liquid is 0.01-0.1U/mu l UDG enzyme, 0.01-0.1U/mu l hot start Taq enzyme, 0.01-0.2U/mu l Taq enzyme antibody protective agent, 100-900 mu M containing dNUTP deoxynucleotide triphosphate;
The working concentration of each component of the PCR enhancer is 1-2M betaine or monohydrate betaine, 20-30 vol% of glycerol and 25-35 mM of tetramethyl ammonium chloride;
The working concentration of each component of the buffer system is 40-60 mmol/l of tris hydrochloride, 15-25 mmol/l of magnesium chloride, 15-25 mmol/l of potassium chloride, 100-200 mmol/l of sodium chloride, 0.1-0.5 vol% of triton and 8-12 vol% of formamide.
A group b streptococcus detection kit comprising the primer probe combination of claim 1 or 2, or comprising the qPCR reaction reagent of claim 3 or 4.
6. The test kit of claim 5, further comprising a nucleic acid extraction reagent; the nucleic acid extraction reagent comprises: pretreatment liquid, lysozyme, lysate, proteinase K, magnetic beads and rinsing liquid;
The lysate comprises: surfactant, polyethylene glycol 4-tert-octylphenyl ester and guanidine hydrochloride.
7. The detection kit according to claim 6, wherein the lysate consists of 0.8wt% of a surfactant, 1.5wt% of polyethylene glycol 4-tert-octylphenyl ester, 4-6mol/L guanidine hydrochloride, and water.
8. The test kit according to claim 6, wherein,
The pretreatment liquid comprises Tris, EDTA, tritonX-100 parts of water;
The rinse solution comprises: tris, na 2 -EDTA, tritonX-100, MOPS, guanidine hydrochloride and water.
9. The test kit according to any one of claims 5 to 8, characterized by comprising a positive control and a negative control;
The positive control is a mixture of CFB plasmid, SIP plasmid and RNaseP plasmid;
The negative control is a human cell line culture.
10. A method for detecting GBS bacteria for non-diagnostic purposes,
Comprising detecting a sample with the primer probe combination of claim 1 or 2;
or comprises detecting the sample with the qPCR reaction reagent of claim 3 or 4;
or comprises detecting the sample with a detection kit according to any one of claims 5 to 9.
CN202410366782.4A 2024-03-27 2024-03-27 PCR-fluorescent probe method kit for optimizing group B streptococcus nucleic acid extraction and amplification detection Pending CN118109620A (en)

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