CN107083434B - Sample pretreatment method for non-diagnosis-purpose fluorescent PCR quantitative detection of group B streptococcus - Google Patents
Sample pretreatment method for non-diagnosis-purpose fluorescent PCR quantitative detection of group B streptococcus Download PDFInfo
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Abstract
The invention relates to the technical field of bacteria detection, in particular to a sample pretreatment method for non-diagnosis-purpose fluorescent PCR quantitative detection of group B streptococcus. The method has the obvious characteristics that bacteria to be detected do not need to be washed from a sample swab to prepare a sample suspension, nucleic acid extraction processes such as high-speed oscillation, boiling and the like do not need to be carried out, group B streptococcus lyase is adopted to directly crack the group B streptococcus in situ on the swab, and the obtained lysate is used as a PCR amplification template after being centrifuged for a short time and is directly used for fluorescence PCR detection. The method has good effect of cracking the streptococcus, has high nucleic acid extraction efficiency, does not need a vibrator, a water bath, an ice bath and other equipment, is beneficial to saving the sample processing cost, can greatly simplify the operation and steps required for extracting the nucleic acid from the swab, and can be used for the subsequent quantitative detection of the streptococcus of the B group by fluorescence PCR.
Description
Technical Field
The invention relates to the technical field of bacteria detection, in particular to a sample pretreatment method for non-diagnosis-purpose fluorescent PCR quantitative detection of group B streptococcus.
Background
Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a opportunistic pathogen that parasitizes the human urogenital tract and lower digestive tract, with a bacterial carrying rate of up to 35% for healthy people, and is not pathogenic for general healthy people. However, the group B streptococcus colonized in the reproductive tract of a parturient can be vertically transmitted to a newborn at the time of delivery, and neonatal septicemia, meningitis, pneumonia and other diseases caused by infection of the group B streptococcus of the newborn have extremely high fatality rate and can cause nervous system sequelae. It is statistically estimated that about 10% to 30% of pregnant women are infected with GBS, of which 40% to 70% are transferred to the neonate during delivery. If the newborn carries this bacteria, about 1% to 3% of them develop early invasive infections, of which 5% lead to death. Group B streptococci have been identified as one of the important pathogens of perinatal maternal and infant infections as early as the 70 th 20 th century. In 2010, the United states disease prevention center sets up 'GBS prevention guideline for perinatal period', and recommends that pregnant women carry out GBS screening at 34-37 weeks of gestation.
At present, the diagnostic methods of group B streptococcus mainly include culture methods, immunological methods, automatic analysis and identification systems of microorganisms and fluorescent PCR methods. The culture method has high specificity and sensitivity, but the clinical detection time is too long (at least 24-48 hours or even longer time), the process is complicated, a special culture medium is required to be used, the method is easily influenced by mixed bacteria, and the method is not suitable for large-scale detection. Immunological methods, such as: latex Agglutination (LA), enzyme linked immunosorbent assay (ELISA), cooperative agglutination (CoA), and Countercurrent Immunoelectrophoresis (CIE), etc., although the specificity can be as high as 95%, the sensitivity is insufficient and the detection rate is low. The U.S. FDA has issued a warning in 1997 that both the false negative and false positive rates of these methods are highly feasible. The automatic microorganism analysis and identification system can comprehensively and automatically identify and detect microorganisms, but is long in time consumption, complicated in identification process, high in requirement on experience of workers, expensive in equipment and high in cost, and is not beneficial to large-scale popularization.
The drawbacks of the above methods are largely overcome by the fluorescent PCR method which has emerged in recent years. The fluorescent quantitative PCR technology is a more sensitive, specific and accurate nucleic acid detection technology developed based on the traditional PCR technology and combined with the spectrum technology. The detection result is accurate, the repeatability is high, the dynamic change of pathogens before and after treatment of a patient and the clinical relation can be dynamically reflected, the problem that the traditional PCR needs post-treatment is avoided in the whole process, and the pollution is reduced. The real-time fluorescence quantitative PCR technology uses a PCR amplification instrument with a fluorescence detection device, the fluorescence detection device can periodically emit exciting light with specific wavelength according to a certain program, collect and detect fluorescence signals, reflect the amplification level of each cycle of PCR in real time by detecting the dynamic change of the fluorescence signals, automatically analyze and obtain an amplification curve through software after the test is finished, and judge the negative and positive results according to the intersection point (namely Ct value) of the amplification curve and a fluorescence threshold line and the shape of the amplification curve; if there is a quantitative reference or standard with a known concentration in the same reaction, a standard curve can be obtained by automatic analysis of software, thereby achieving a fixed value (i.e. quantitative detection) for an unknown sample. Compared with the traditional PCR, the method adds a probe with two ends respectively marked with a fluorescent reporter group and a quenching group in a reaction system. When the probe structure is complete, the fluorescence energy emitted by the fluorescence reporter group is absorbed by the quenching group, and the quenching effect is presented; if a target sequence exists in the amplification process, the probe molecules are gradually hydrolyzed and cut off by Taq enzyme along with the extension of the target fragment, the fluorescent reporter group and the quenching group are dissociated with each other, the fluorescent energy transfer effect between the fluorescent reporter group and the quenching group is blocked, and a fluorescent signal emitted by the fluorescent reporter group is collected by a fluorescent detection device. As amplification proceeds, the fluorescent signal exhibits a linear increase as the target fragment is amplified. After the test is finished, the data can be automatically analyzed through software carried by the fluorescence PCR instrument, and negative and positive results and fixed value results of sample concentration can be obtained, so that the technology gradually replaces the traditional PCR method in the detection and quantitative analysis of target polynucleotide samples, and is widely applied. Studies have shown that real-time fluorescent PCR detection methods and standard bacterial culture methods are more than 90% sensitive and specific to GBS detection, and have been approved by the U.S. Food and Drug Administration (FDA) and applied to clinical screening.
Before PCR detection, nucleic acid is extracted from clinical samples and used as a template for amplification. In the current GBS screening for pregnant women, a sterile swab is generally used for collecting a sample, materials such as terylene, fibers or nylon and the like are used on a swab head, and a swab handle is made of a plastic material. During sampling, the swab head is placed in the genital tract and rectum respectively to rotate to collect secretion. Sample processing of the swab is required prior to real-time fluorescent PCR amplification. The method used at present is that the swab after sampling is put into the cleaning liquid, a vibrator is used for high-speed oscillation for several minutes, bacteria are released from the swab head to prepare a specimen suspension, and then subsequent processes such as nucleic acid extraction and the like are carried out, such as specimen suspension concentration, washing, adding lysate, boiling, high-speed centrifugation and the like. Finally, taking a certain supernatant as a PCR amplification template. In practical clinical application, the method has the following defects: (1) the nucleic acid extraction process is complex and requires a plurality of instruments and equipment; (2) the sample treatment consumes long time, and DNA loss is serious after the steps of enrichment, cleaning, boiling and cracking, high-speed centrifugation and the like when the sample is treated; (3) in the steps of high-temperature heating and the like involved in sample processing, if the tube cover is broken open, the risks of aerosol pollution, sample loss and the like exist.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a sample pretreatment method for non-diagnosis-purpose fluorescent PCR quantitative detection of group B streptococcus, the method adopts group B streptococcus lyase to directly crack the group B streptococcus on a swab in situ, and the obtained lysate can be directly used for fluorescent PCR detection, thereby solving the problems of complicated sample treatment process, long time consumption, sample pollution risk and the like in the prior art.
The technical scheme of the invention is realized as follows:
a sample swab pretreatment method for quantitative detection of group B streptococcus through fluorescence PCR comprises the steps of dropwise adding a group B streptococcus lyase liquid onto a swab to be detected, standing at room temperature for 5-30min, directly extruding a lysate from the swab, centrifuging the lysate, and taking a proper amount of supernatant as a fluorescence PCR amplification template.
Further, the group B streptococcus lyase solution comprises GBS-PlySb enzyme solution and group B streptococcus lyase buffer solution.
Further, the amino acid sequence of the GBS-PlySb enzyme is:
MATYQEYKSRSNGNAYDIDGSFGAQCWDGYADYCKYLGLPYANCTNTGYARDIWEQRHENGILNYFDEVEVMQAGDVAIFMVVDGVTPYSHVAIFDSDAGGGYGWFLGQNQGGANGAYNIVKIPYSATYPTAFRPKVFKNAVTVTGNIGLNKGDYFIDVSAYQQADLTTTCQQAGTTKTIGSSRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVIIDVNGYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK。
furthermore, the GBS-PlySb enzyme concentration in the GBS-PlySb enzyme solution is 20-200 mu g/mL.
Further, the buffer solution of the group B streptococcus lyase comprises one or more of Tris buffer solution, glycine-NaOH buffer solution, HEPES buffer solution and MES buffer solution.
Further, the concentration of the group B streptococcus lyase buffer solution is 10-100 mmol/L, and the pH value is 6-8.
An application of a sample swab pretreatment method for quantitatively detecting group B streptococcus by fluorescence PCR is applied to the preparation of a fluorescence quantitative PCR detection kit for detecting group B streptococcus in the reproductive tract or intestinal tract of a pregnant woman.
Has the advantages that: compared with the prior art, the method adopts the group B streptococcus lyase to directly crack the group B streptococcus on the swab in situ, and the obtained lysate can be directly used for fluorescence PCR detection, so that the problems of complicated sample treatment process, long consumed time, sample pollution risk and the like in the prior art are solved, and when the method is adopted to quantitatively detect the group B streptococcus, the streptococcus cracking effect is better, and the nucleic acid extraction efficiency is higher.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the method for quantitative detection of group B streptococcus by fluorescence PCR according to the present invention;
FIG. 2 is a comparison graph of the results of fluorescence quantitative PCR detection using lysates obtained after samples are treated by the method and the commercial kit of the present invention, wherein curve 1 is an amplification curve obtained by in situ lysis of bacteria on a swab, and curve 2 is an amplification curve obtained by treating samples with the commercial kit.
Detailed Description
The following examples are intended to be merely illustrative of certain embodiments of the present invention and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention.
Example 1: the specific implementation steps for carrying out fluorescence PCR quantitative detection on group B streptococcus by adopting the method of the invention
As shown in figure 1, firstly, sampling by using a swab, dripping a group B streptococcus lyase solution on the swab to be detected, standing at room temperature for 5-30min, directly extruding the lysate from the swab (for example, pressing a swab head on the wall of a centrifugal tube), centrifuging the extruded lysate, finally taking a proper amount of supernatant as a PCR amplification template to a PCR system, and collecting a fluorescent signal. The method is characterized in that the lysate is directly dripped on the swab head for in-situ lysis without washing the bacteria to be detected from the swab to prepare a specimen suspension. After the lysis is finished, extruding a certain amount of lysate from the swab head, centrifuging for a short time, and taking a certain amount of supernatant as a template for PCR amplification according to the requirements of the fluorescent quantitative PCR kit. The method greatly simplifies the operations and steps required to extract nucleic acids from a swab.
In order to illustrate the advantages of the method, in the following specific examples, a clinically isolated streptococcus agalactiae was used as a simulated sample, a commercial B-cluster streptococcus nucleic acid detection kit (fluorescence PCR method) (taipu biosciences (china) ltd) was used as a control, and experiments were performed according to the instructions thereof, and at the same time, the method of the present invention was used, and a streptococcus lyase kit capable of rapidly cleaving streptococcus at normal temperature (the kit was a product of wuhansai seiki biotechnology ltd, product number: cleavage 01) was used to perform comparative tests. For the obtained lysate template, a PCR reagent in a commercial B cluster streptococcus nucleic acid detection kit (fluorescence PCR method) (Tepu bioscience (China) Co., Ltd.) is adopted for amplification detection.
Preparation of GBS-PlySb enzyme
Preparation of catalytic Domain of PlyGBS, cell wall binding Domain of PlySs2
The lyase PlyGBS and the lyase PlySs2 gene fragments (Nanjing Kingsri Biotechnology Co., Ltd.) were synthesized in their full sequence, and the synthetic sequences were packaged in pUC57 plasmid. The sequence of catalytic domain GBS180 of PlyGBS is amplified by taking plyGBS gene as a template and GBS180-F/GBS180-R as a primer, and the cell wall binding domain of PlySs2 is amplified by taking plySs2 gene as a template and PlySb-F/PlySb-R as a primer. The primers and restriction site information used in the cloning construction were as follows:
GBS180-F:5-TTAACCATGGGCATGGCTACCTACCAGG-3
GBS180-R:5-TATAGGATCCGATCGTTTTGGTCGTGC-3
PlySb-F:5-TATAGGATCCTCTCGTTCCTATCGCGAG-3
PlySb-R:5-TATACTCGAGTTTAAATGTACCCCAAG-3
the GBS180-F primer is underlined the restriction enzyme NcoI site, the GBS180-R and PlySb-F primers are underlined the restriction enzyme BamHI site, and the PlySb-R primer is underlined the restriction enzyme XhoI site.
The procedure for PCR amplification of gene fragments was as follows: taking 2 mu L of gene as a template, adding 1 mu g of primers respectively for PCR amplification, wherein the PCR reaction conditions are as follows: (1) pre-denaturation at 94 ℃ for 5 min; (2) denaturation at 94 ℃ for 30sec, 62 ℃, 45sec, 72 ℃, 45sec, 30 cycles; (3) extension at 72 ℃ for 10 min.
2. Soluble expression of GBS-PlySb protein
The above fragments were digested with the corresponding enzymes and ligated into pET28b (+) vector digested with NcoI and XhoI to obtain expression vector pET28b-GBS-PlySb for GBS-PlySb. It was then transformed into E.coli BL21(DE 3). GBS180-F/PlySb-R is used as a primer to carry out PCR verification on the transformant, and the GBS-PlySb gene with the full length can be obtained through amplification.
Expression purification of GBS-PlySb
The expression strain BL21(DE3)/pET28b-GBS-PlySb was subjected to low-temperature induction with 2mM IPTG for expression. Collecting the thallus, carrying out ultrasonication, taking the supernatant, passing the supernatant through a Ni affinity column, collecting a 250mM elution peak, and dialyzing the eluate in PBS overnight.
The dialyzed protein can be further purified by ion exchange resin, passed through HiTrap Q Sepharose FFcolumn (GEHealthcare), and the column effluent collected. The column effluent was passed through a HiTrap SP Sepharose FF column and then eluted with a 1M NaCl gradient, and the eluted peaks were collected by fractions. Mixing the active tubes, and dialyzing in PBS overnight to obtain purified enzyme solution. The enzyme solution can be further added with a freezing protective agent to prepare freeze-dried powder through freeze drying, so that the enzyme solution is convenient to store at low temperature and normal temperature for a long time and can keep the activity of the enzyme.
Reagent and swab sample preparation
Adding the resuscitation solution into a streptococcus freeze-dried powder bottle according to the instruction of a streptococcus lyase kit, and mixing for later use;
putting the lyase solution into a refrigerator at 4 ℃ for later use;
following commercial kit instructions, 10x concentrated wash was taken out with sterile purified water at a ratio of 1: 9 (volume ratio) and putting the diluted solution into a refrigerator at 4 ℃ for later use;
after calculating the number n of reaction tubes to be performed (the number of samples plus negative and positive controls), taking out the GBS-PCR reaction liquid and enzyme, and the like, adding nx44.3 mu L of GBS-PCR reaction liquid, nx0.5 mu L of Taq enzyme liquid and nx0.2 mu L UNG into a centrifuge tube, uniformly oscillating, instantly centrifuging, subpackaging into n PCR reaction tubes, each tube being 45 mu L, covering the tube cover, transferring to a sample adding area, and keeping out of the sun and placing in a refrigerator at 4 ℃ for later use.
Simulated sample preparation and swab sampling: taking a small amount of preserved agalactia streptococcus bacteria liquid, inoculating the agalactia streptococcus bacteria liquid into an LB culture medium on a super clean bench for overnight culture at 37 ℃, taking 5mL of the agalactia streptococcus bacteria liquid into a 15mL centrifugal tube, centrifuging the agalactia streptococcus bacteria liquid for 5min at 10000r/min, removing supernatant, taking 5mL of cleaning solution in a tylosin kit for resuspending the bacteria, and using the bacteria liquid diluted by 1000 times of the cleaning solution as a simulated secretion. 2 sterile swabs (disposable swabs (female swabs), Jiangsu health and medical supplies, Inc.) are taken, swab heads are respectively immersed into the bacteria liquid for 5s and taken out, and the collected sterile swabs are put back into a sterile swab sleeve to finish sampling.
Thirdly, sample processing:
the method A comprises the following steps: following the protocol in the commercial kit instructions.
Adding 1mL of cleaning solution into the swab sleeve, and shaking at high speed for 2min by using an oscillator to prepare a specimen suspension. All samples were taken out and placed in a 1.5mL centrifuge tube, centrifuged at 13000r/min for 5min to remove supernatant, and 50. mu.L of washing solution was added for resuspension. The extract solids from 1 tube kit were added to the sample tube and vortexed at high speed for 5min using a high-speed vortexer (Vortex-Genie, usa). And (3) carrying out water bath on the sample tube after shaking for 2min at 95 ℃, immediately carrying out ice bath for 2-5min, centrifuging for 1min at 13000r/min, and taking 5 mu L of supernate to carry out amplification in a PCR system.
The method B comprises the following steps: in situ lysis of group B streptococci on swabs for fluorescent PCR detection
100 μ L of group streptococcal lyase solution GBS-PlySb enzyme solution with the concentration of 20 μ g/mL is dripped on the swab head, and the swab is put back into the swab sleeve and is kept stand for 10min at room temperature. The swab was removed from the cannula, the swab tip was inserted into a 1mL sterile plastic pipette tip, and approximately 20-50. mu.L of lysate was squeezed out into a 1.5mL sterile centrifuge tube. Lysate was centrifuged at 13000r/min for 1min, and 5. mu.L of supernatant was taken for PCR amplification.
Fourthly, fluorescent real-time quantitative PCR detection:
1. setting a PCR program:
37℃，2min；
94℃，2min；
and (4) circulating for 10 times: 94 ℃, 20 s; 45s at 55 ℃;
and (3) circulating for 30 times: 94 ℃, 20 s; 55 ℃ for 45 s.
A Bio-Rad CFX Connect fluorescence quantitative PCR instrument is adopted, and GBS detects that fluorescein is FAM. The fluorescent signal was collected at the fourth stage at 55 ℃ for 45s, and the resulting amplification curve is shown in FIG. 2.
In this example, the Ct value of the amplification curve obtained by the commercial kit method was 16.20, and the Ct value of the amplification curve obtained by the in situ lysis of group B streptococci on cotton swabs was 12.8. From the qualitative result, both obtained positive results, and the interpretation of the results was not affected. However, in terms of numerical value, the extraction efficiency of the method for in situ lysis of group B streptococcus on a swab is smaller than the Ct value of the commercial kit method, and the lysis effect is relatively good, because bacteria need to be released from the swab when a sample is processed by the current commercial kit method, the release process is not 100%, and in the subsequent centrifugal concentration and other processes of a sample suspension, a part of bacteria may be lost, so that the number of finally obtained nucleic acid templates is small. In the method, the bacteria are not required to be released from the swab and are directly cracked by the added group B streptococcus lyase liquid in situ, the bacteria on the swab can be cracked by 100 percent, and the amount of the released nucleic acid is larger without a subsequent treatment process. More importantly, compared with the sample processing method of the commercial kit, the method for in-situ cracking the swab has the advantages of shortened steps, simple and convenient operation, and only about 12min of time, while the commercial method needs about 25 min. In addition, in the whole treatment process, the method does not need equipment such as a vibrator, a dry bath kettle, an ice bath and the like, and is favorable for saving the sample treatment cost.
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.
Claims (4)
1. A sample swab pretreatment method for non-diagnostic fluorescent PCR quantitative detection of group B streptococcus is characterized by comprising the following steps: dropwise adding group B streptococcus lyase liquid onto a swab to be detected, standing at room temperature for 5-30min, directly extruding lysate from the swab, centrifuging the lysate, and taking a proper amount of supernatant as a fluorescent PCR amplification template;
wherein the group B streptococcus lyase solution comprises GBS-PlySb enzyme solution and group B streptococcus lyase buffer solution; the amino acid sequence of the GBS-PlySb enzyme is as follows:
MATYQEYKSRSNGNAYDIDGSFGAQCWDGYADYCKYLGLPYANCTNTGYARDIWEQRHENGILNYFDEVEVMQAGDVAIFMVVDGVTPYSHVAIFDSDAGGGYGWFLGQNQGGANGAYNIVKIPYSATYPTAFRPKVFKNAVTVTGNIGLNKGDYFIDVSAYQQADLTTTCQQAGTTKTIGSSRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVIIDVNGYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK。
2. the method for pre-treating a sample swab for the quantitative detection of group B streptococci by fluorescent PCR for non-diagnostic purposes as claimed in claim 1, wherein: the GBS-PlySb enzyme concentration in the GBS-PlySb enzyme solution is 20-200 mu g/mL.
3. The method for pre-treating a sample swab for the quantitative detection of group B streptococci by fluorescent PCR for non-diagnostic purposes as claimed in claim 1, wherein: the group B streptococcus lyase buffer solution comprises one or more of Tris buffer solution, glycine-NaOH buffer solution, HEPES buffer solution and MES buffer solution.
4. The method for pre-treating a sample swab for the quantitative detection of group B streptococci by fluorescent PCR for non-diagnostic purposes as claimed in claim 1, wherein: the concentration of the group B streptococcus lyase buffer solution is 10-100 mmol/L, and the pH value is 6-8.
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| WO2011065854A1 (en) * | 2009-11-24 | 2011-06-03 | Technophage, Investigação E Desenvolvimento Em Biotecnologia, Sa | Enterococcal phage peptides and methods of use thereof |
| CN103409509A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Group B streptococcus fluorescence PCR detection kit |
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