CN111850143B - GBS DNA nucleic acid detection method - Google Patents
GBS DNA nucleic acid detection method Download PDFInfo
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- CN111850143B CN111850143B CN202010317546.5A CN202010317546A CN111850143B CN 111850143 B CN111850143 B CN 111850143B CN 202010317546 A CN202010317546 A CN 202010317546A CN 111850143 B CN111850143 B CN 111850143B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a GBS DNA nucleic acid detection method, which comprises the following steps: adding physiological saline into a sterile terylene swab tube, and uniformly mixing to prepare a specimen suspension; centrifuging at 13000rpm for 10min, and discarding the supernatant; adding absolute ethanol into the precipitate, mixing, centrifuging at 10000-13000rpm for 5-10min, and discarding the supernatant; adding sterile normal saline into the precipitate, mixing uniformly, centrifuging at 13000rpm for 5min, and discarding the supernatant; adding DNA extracting solution into the precipitate, mixing uniformly, and heating; centrifuging at 13000rpm for 10min, and collecting supernatant; adding a sample to be detected into a PCR reaction tube and then performing instantaneous centrifugation; and (3) placing the PCR reaction tube in a real-time fluorescent quantitative PCR instrument for amplification reaction, and judging the result according to the amplification curve. The method has simple steps and easy popularization, improves the detection rate by about 2.4 percent after the method is used, is more favorable for clinical medication guidance, and has wide application prospect.
Description
Technical Field
The invention relates to the field of nucleic acid detection methods, in particular to a GBS DNA nucleic acid detection method.
Background
The group B streptococcus is facultative anaerobic gram-positive streptococcus which normally inhabits the vagina and rectum, the bacteria carrying rate of normal women reaches about 30 percent, the group B streptococcus belongs to conditional pathogenic bacteria, pregnant women can infect and spread to newborns through birth canals to cause newborn septicemia, meningitis, pneumonia and the like, the death rate is extremely high, and the group B streptococcus is one of the most main pathogenic bacteria causing newborn infection at present. The patient can gather the sample censorship of two sampling positions, and the position of taking has rectum and vagina, often can gather more excrement and urine/secretion, can lead to the experimental result to receive the suppression, does not have the amplification curve.
Disclosure of Invention
The present invention provides a method for detecting GBS DNA nucleic acid, which solves the above technical problems.
The invention is realized according to the following technical scheme.
A method for detecting GBS DNA nucleic acid comprising the steps of:
a. adding physiological saline into a sterile terylene swab tube, and uniformly mixing to prepare a specimen suspension;
b. taking out all samples, centrifuging at 13000rpm for 10min, and discarding the supernatant;
c. adding absolute ethyl alcohol into the sediment obtained in the step b, uniformly mixing, centrifuging at 10000-13000rpm for 5-10min, and discarding the supernatant;
d. adding sterile normal saline into the sediment obtained in the step c, uniformly mixing, centrifuging at 13000rpm for 5-10min, and discarding the supernatant;
e. d, adding the DNA extracting solution into the precipitate obtained in the step d, uniformly mixing, and heating;
f.13000rpm centrifugation for 10min, suction of the supernatant for preservation;
g. f, adding the sample to be detected obtained in the step f into a PCR reaction tube and then performing instantaneous centrifugation;
h. and (3) placing the PCR reaction tube in a real-time fluorescent quantitative PCR instrument for amplification reaction, and judging the result according to an amplification curve.
Further, in step c, the centrifugation condition was 13000rpm for 5min.
Further, in the step e, the heating treatment condition is 100 +/-5 ℃ for 10-12min.
Further, in step g, the sample to be detected is added into the PCR reaction tube, and the extracted GBS positive control and the extracted blank control are also added into the PCR reaction tube respectively.
Further, the extraction method of the GBS positive control comprises the following steps: taking a GBS positive control, centrifuging at 13000rpm for 10 minutes, and removing the supernatant; adding DNA extracting solution into the precipitate, fully and uniformly mixing, and heating for 10 minutes at 100 ℃; centrifuging at 13000rpm for 10 minutes, sucking the supernatant and storing; the extraction method of the blank reference substance is the same as that of the GBS positive reference substance.
Further, in the step h, the reaction conditions of the real-time fluorescence quantitative PCR are as follows: UNG reaction: 2min at 50 ℃; b. pre-deformation: 5min at 95 ℃; c, PCR: 45 cycles of 95 ℃ for 15s to 60 ℃ for 35 s; and respectively detecting fluorescence signals of FAM and HEX channels at 60 ℃, and selecting 40uL of the reaction system.
The present invention obtains the following advantageous effects.
The GBS DNA nucleic acid detection method has simple steps and easy popularization, improves the detection rate by about 2.4 percent after using the method, is more beneficial to clinical medication guidance, and has wide application prospect.
Drawings
FIG. 1 is a graph showing the real-time fluorescent quantitative PCR amplification curve (sample is negative) of the method of the present invention;
FIG. 2 is a graph showing the real-time fluorescence quantitative PCR amplification curve of the present invention (positive sample);
FIG. 3 is a graph showing the real-time fluorescence quantitative PCR amplification curve of the present invention (the sample needs to be retested);
FIG. 4 is a graph showing the real-time fluorescent quantitative PCR amplification curve of the present invention (negative after retesting of the sample);
FIG. 5 is a graph of a prior art method real-time fluorescent quantitative PCR amplification.
Detailed Description
The invention is further explained below with reference to the drawings and the examples.
Examples
A GBS DNA nucleic acid detection method, comprising the steps of:
1. 1mL of physiological saline is added into a sterile terylene swab tube, and the mixture is shaken and mixed evenly by a vortex oscillator to prepare specimen suspension.
2. All samples were taken out and placed in a 1.5mL centrifuge tube, centrifuged at 13000rpm for 10min and the supernatant discarded.
3. The precipitate was mixed with 1mL of absolute ethanol, centrifuged at 13000rpm for 5min and the supernatant discarded.
4. The precipitate was mixed with 1mL of sterile physiological saline, centrifuged at 13000rpm for 5min and the supernatant discarded.
5. Adding 50uL DNA extractive solution (group B streptococcus (GBS) nucleic acid detection kit/Boerchen (Beijing) science and technology Co., ltd.) into the precipitate, shaking thoroughly, mixing, and heating at 100 deg.C for 10min.
5363 and centrifuging at 6.13000rpm for 10min, sucking supernatant to a 1.5mL centrifuge tube for preservation, and finishing DNA advance.
7 adding 5uL of sample to be detected, extracted positive reference substance and blank reference substance (group B streptococcus (GBS) nucleic acid detection kit/Boerchen (Beijing) science and technology Co., ltd.) into the prepared PCR reaction tube, covering the tube cover tightly, and centrifuging instantly.
Extracting a GBS positive control: 50uL of GBS positive control was added to a 1.5uL centrifuge tube (filled to 1mL with sterile physiological saline), centrifuged at 13000rpm for 10 minutes, and the supernatant was discarded. Adding 50uLDNA extracting solution into the precipitate, fully and uniformly mixing, heating at 100 ℃ for 10 minutes, centrifuging at 13000rpm for 10 minutes, sucking supernatant to a 1.5mL centrifuge tube for storage, and finishing DNA extraction.
Blank control extraction: extracting with GBS positive control.
8. Transfer to the amplification zone through the sample delivery window.
9. Carefully placing the PCR reaction tube to be detected in an ABI 7500 real-time fluorescent quantitative PCR instrument for amplification reaction, and judging the result according to the amplification curve.
The reaction conditions of the real-time fluorescent quantitative PCR are as follows: UNG reaction: 2min at 50 ℃; b. pre-deformation: 5min at 95 ℃; c, PCR: (95 ℃ for 15 seconds to 60 ℃ for 35 seconds) for 45 cycles; and (3) respectively detecting fluorescence signals of FAM and HEX channels at 60 ℃, and selecting a reaction system of 40uL.
10. After the machine is operated, the result has an amplification curve, and the result is judged to be negative.
The normal amplification curve is a standard sigmoidal amplification curve.
And (5) judging a result:
1. and (4) judging a negative result: the FAM channel has no Ct value to display, and the HEX channel signal is normal (Ct is less than or equal to 35.00), the sample is negative, as shown in figure 1 (BZL 0001 has no negative CT value);
2. and (3) judging a positive result:
2.1 if the FAM channel Ct of the sample is less than or equal to 38.00, the sample is determined to be positive, as shown in FIG. 2 (the BZL00022 amplification curve is a standard sigmoid curve, and the CT value =29 positive);
2.2 if Ct of the sample is more than 38.00 and less than or equal to 45.00, the sample is recommended to be retested. If the retest result is still more than 38.00 and Ct is less than or equal to 45.00 or Ct is less than or equal to 38.00, the result is judged to be positive; if the Ct is not displayed and the HEX channel signal is normal (Ct is less than or equal to 35.00), the result is judged to be negative. FIG. 3 (BZL 0020 CT =40 gray zone, repeat test required), FIG. 4 (no CT value after FBZL0020 repeat test, normal amplification curve, negative result).
The preparation process of the sample to be detected for detecting the group B streptococcus nucleic acid is as follows (the prior art):
1. 1mL of physiological saline is added into a sterile terylene swab tube, and a specimen suspension is prepared by shaking and uniformly mixing the saline with a vortex oscillator.
2. All samples were taken out and put into a 1.5mL centrifuge tube, centrifuged at 13000rpm for 10min and the supernatant discarded.
3. The precipitate was mixed with 1mL of sterile physiological saline, centrifuged at 13000rpm for 5min and the supernatant discarded.
4. Adding 50uL of DNA extract into the precipitate, shaking and mixing uniformly, and heating at 100 ℃ for 10min.
5363 and centrifuging at 5.13000rpm for 10min, sucking supernatant to a 1.5mL centrifuge tube for preservation, and finishing DNA advance.
6. 5uL of sample to be tested, positive reference substance and blank reference substance are distributed and added into the prepared PCR reaction tube, and the tube cover is covered tightly and then is centrifuged instantly.
7. Transfer to the amplification zone through the sample delivery window.
8. Carefully placing the PCR reaction tube to be detected in an ABI 7500 real-time fluorescent quantitative PCR instrument for amplification reaction, judging the result according to the amplification curve, and issuing a bill (as shown in figure 5).
As shown in FIG. 5, when the prior art method is used for operation treatment, the amplification curve has no peak, no amplification result exists, and the reported result can only be processed for singleness rejection, so that the detection rate is reduced. The technical scheme of the application can generate an amplification curve by processing the same sample, and the detection rate of the GBS DNA of the sample is improved.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (6)
1. A method for detecting GBS DNA nucleic acid of non-diagnostic interest, characterized in that: the method comprises the following steps:
a. adding physiological saline into a sterile terylene swab tube, and uniformly mixing to prepare a specimen suspension;
b. taking out all samples, centrifuging at 13000rpm for 10min, and discarding the supernatant;
c. adding absolute ethyl alcohol into the sediment obtained in the step b, uniformly mixing, centrifuging at 10000-13000rpm for 5-10min, and discarding the supernatant;
d. adding sterile normal saline into the sediment obtained in the step c, uniformly mixing, centrifuging at 13000rpm for 5-10min, and discarding the supernatant;
e. d, adding the DNA extracting solution into the precipitate obtained in the step d, uniformly mixing, and heating;
f.13000rpm centrifugation for 10min, suction of the supernatant for preservation;
g. f, adding the sample to be detected obtained in the step f into a PCR reaction tube and then performing instantaneous centrifugation;
h. and (3) placing the PCR reaction tube in a real-time fluorescent quantitative PCR instrument for amplification reaction, and judging the result according to the amplification curve.
2. The method of claim 1 for the detection of GBS DNA nucleic acid of non-diagnostic interest, wherein: in step c, centrifugation is carried out for 5min at 13000 rpm.
3. The method of claim 1 for the detection of GBS DNA nucleic acid of non-diagnostic interest, wherein: in the step e, the heating treatment condition is 100 +/-5 ℃ for 10-12min.
4. The method of claim 1 for the detection of GBS DNA nucleic acid of non-diagnostic interest, wherein: and step g, adding the sample to be detected into the PCR reaction tube, and simultaneously respectively adding the extracted GBS positive reference substance and the blank reference substance into the PCR reaction tube.
5. The method of claim 4 for detecting GBS DNA nucleic acid of non-diagnostic interest, wherein: the extraction method of the GBS positive reference substance comprises the following steps: taking a GBS positive control, centrifuging at 13000rpm for 10 minutes, and removing the supernatant; adding DNA extracting solution into the precipitate, fully and uniformly mixing, and heating for 10 minutes at 100 ℃; centrifuging at 13000rpm for 10 minutes, sucking the supernatant and storing; the extraction method of the blank reference substance is the same as that of the GBS positive reference substance.
6. The method of claim 1 for the detection of GBS DNA nucleic acid of non-diagnostic interest, wherein: in the step h, the reaction conditions of the real-time fluorescence quantitative PCR are as follows: UNG reaction: 2min at 50 ℃; b. pre-deformation: 5min at 95 ℃; c, PCR: 45 cycles of 95 ℃ for 15s to 60 ℃ for 35 s; and (3) respectively detecting fluorescence signals of FAM and HEX channels at 60 ℃, and selecting a reaction system of 40uL.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557443A (en) * | 2017-10-27 | 2018-01-09 | 广州赛哲生物科技股份有限公司 | Group B streptococcus fluorescence quantitative PCR detection kit |
| CN108841975A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | One kind is for detecting the streptococcic nucleotide sequence group of B race, kit and method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557443A (en) * | 2017-10-27 | 2018-01-09 | 广州赛哲生物科技股份有限公司 | Group B streptococcus fluorescence quantitative PCR detection kit |
| CN108841975A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | One kind is for detecting the streptococcic nucleotide sequence group of B race, kit and method |
Non-Patent Citations (1)
| Title |
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| 肠道菌群结构多样性初筛研究;杜彩贺等;《南京晓庄学院学报》;20100531(第3期);第68页1.1.2，第69页左栏倒数第1段、右栏第1段 * |
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