CN117587109A - PCR detection method and application thereof - Google Patents

PCR detection method and application thereof Download PDF

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CN117587109A
CN117587109A CN202410077552.6A CN202410077552A CN117587109A CN 117587109 A CN117587109 A CN 117587109A CN 202410077552 A CN202410077552 A CN 202410077552A CN 117587109 A CN117587109 A CN 117587109A
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pcr
dna
sample
solution
ethanol
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张馨月
刘丽苹
路放
宋丽影
荣丽平
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Jilin Jinyu Medical Laboratory Co ltd
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Abstract

The invention discloses a PCR detection method and application thereof. The method comprises the steps of using a washing solution to treat a sample to be detected to extract DNA, heating and centrifuging to obtain target DNA, hybridizing an amplified product with microspheres after PCR amplifying the target DNA, and carrying out fluorescence value detection on hybridization; the washing liquid comprises a combination of a Nacl solution and ethanol, wherein the Nacl solution comprises 80-90% by mass of Nacl; the mass percentage of the ethanol is 70-75%. The PCR detection method can effectively improve the sample detection rate, ensure the stability and accuracy of the PCR detection result, reduce the inhibition of internal standards caused by different reasons in the experimental process, and reduce the laboratory sheet withdrawal rate.

Description

PCR detection method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a PCR detection method and application thereof.
Background
Cervical cancer is a high-incidence malignant tumor of women, and has serious influence on the life safety of women, and related reports show that the worldwide number of new cervical cancer patients is more than 50 tens of thousands and the death number is more than 27 tens of thousands each year. HPV infection is a major factor in cervical cancer, and studies indicate that 99.7% of cervical cancer patients are infected with HPV. Men infected with HPV are mainly asymptomatic carriers, which are latent vectors of HPV viruses, probably because of the large amount of prepuce dirt and secretions contained in the prepuce and the local environment favorable for HPV growth and reproduction.
Group B Streptococcus (GBS) is the most common group of pathogenic streptococci, also a more dangerous one, also known as streptococcus agalactiae, and is a beta hemolytic aerobic gram positive streptococcus. Is generally non-pathogenic, but can be manifested as bacteremia and urinary system infection when the pregnant woman is infected. GBS infection in pregnant women is closely related to the occurrence of diseases such as premature birth, puerperal infection, neonatal septicemia, etc. The acid-base balance in the vagina of the pregnant woman is broken, so that the vagina has inflammatory symptoms, the fetal membranes are damaged, and finally the fetal membranes are at risk of early rupture. GBS screening of pregnant women in the late 35-37 weeks of pregnancy is taken as a spare examination item for prenatal examination in China published in 2018. The pregnant women infected by GBS need to be treated in time after being diagnosed so as to reduce premature rupture of fetal membranes and risk of intrauterine infection, and the GBS can cause neonatal meningitis, endocarditis, soft tissue infection or osteomyelitis in the delivery process, so that the life health of the neonate is directly affected.
Sexually Transmitted Diseases (STDs) are a type of urogenital tract infectious diseases with sexual contact as a main transmission mode, and Neisseria Gonorrhoeae (NG), chlamydia Trachomatis (CT) and Ureaplasma Urealyticum (UU) are the most common STD pathogens (three items for short, venereal disease), and have high infection rate and recurrence rate, and the incidence rate is also increasing in recent years. In recent years, the research on the infection condition of UU in three related venereal diseases in PROM patients is increased, the research on common infection strains of other female genital tracts is gradually brought into the public view, UU is used as a prokaryotic microorganism, the size of UU is between bacteria and viruses, the UU can be quickly bred and reproduced in an artificial culture environment, an infected person can be infected through direct contact or clothing indirect contact, the UU can be infected through the female genital tracts to cause intrauterine infection, and meanwhile, the UU can also vertically infect fetuses through placenta, and at present, a plurality of researches prove that the UU can cause male infertility, premature birth and abortion and can also participate in causing pulmonary dysplasia of newborns to cause related bad pregnancy ending. Clinically, the mortality rate is low, but the physical and mental health of a patient is seriously affected, and male NG and CT infection can cause urethritis, prostatitis, epididymitis and infertility, and can cause cervicitis, pelvic inflammatory disease, infertility and the like for females.
Polymerase Chain Reaction (PCR) technology has been widely used in various fields of medicine, genetics, archaeology, etc. as an important method for scientific research and has shown a broad application prospect. PCR is a molecular biological technique for amplifying specific DNA fragments, and can be regarded as specific DNA replication in vitro, and the greatest feature of PCR is that it can greatly increase trace amounts of DNA. With the discovery and application of thermostable DNA polymerase in thermotolerant bacteria, the efficiency of PCR is greatly improved and the development of the PCR automation technology is promoted, the PCR is to utilize the fact that DNA becomes single-stranded at a high temperature of 95 ℃ in vitro, the primer and the single-stranded are combined according to the base complementary pairing principle at a low temperature (usually about 60 ℃), the temperature is adjusted to the optimal reaction temperature (about 72 ℃) of the DNA polymerase, and the DNA polymerase synthesizes complementary strand along the direction from phosphoric acid to pentose. The PCR instrument based on polymerase is actually a temperature control device, and can well control the denaturation temperature, the renaturation temperature and the extension temperature. However, the internal standard of the sample is often abnormal during the PCR experiment.
Therefore, it is necessary to provide a pretreatment method for PCR detection experiments, which ensures the accuracy and stability of the PCR experiments.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the PCR detection method and the application thereof, which can reduce the abnormal condition of the internal standard caused by different reasons in the experimental process and ensure the accuracy and the stability of the PCR experiment.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of PCR detection, the method comprising: treating a sample to be detected by using a washing solution to extract DNA, heating and centrifuging to obtain target DNA, hybridizing an amplified product with microspheres after PCR amplifying the target DNA, and carrying out fluorescence value detection on hybridization; the washing liquid comprises a combination of a Nacl solution and ethanol, wherein the Nacl solution comprises 80-90% by mass of Nacl; the mass percentage of the ethanol is 70-75%.
The PCR detection method can effectively improve the sample detection rate, ensure the stability and accuracy of the PCR detection result, reduce the abnormal condition of the internal standard caused by different reasons in the experimental process, be applied to the experimental process and reduce the laboratory sheet withdrawal rate.
Preferably, the mass percentage of the brine is 80% -90%.
Specific point values in the above 80% -90% may be selected from 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% and the like.
Specific point values of 70% -75% can be selected from 70%, 71%, 72%, 73%, 74%, 75% and the like.
Preferably, the sample to be tested comprises any one or a combination of at least two of human papillomavirus HPV-DNA, group B streptococcus qualitative GBS-DNA or a qualitative sample of a venereal disease.
Preferably, the washing liquid further comprises isopropyl alcohol; the volume ratio of the ethanol to the isopropanol in the washing liquid is (0.5-3): 1.
Specific point values in the above 0.5-3 may be selected from 0.5, 0.6, 0.7, 1.0, 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 3, etc.
Preferably, the washing solution further comprises a buffer solution.
Preferably, the buffer comprises KCl solution or MgCl 2 A solution; the pH of the buffer is 4-6.
Specific point values of 4 to 6 may be selected from 4, 5, 6, etc.
Preferably, the volume ratio of the NaCl solution, the alcohol and the buffer solution in the washing liquid is 1 (1-3): 1.
Specific point values in the above 1 to 3 may be 1, 2, 3, etc.
In a second aspect, the invention provides the use of the PCR detection method of the first aspect in SSR-PCR, ISSR-PCR, RACE-PCR, touchdown PCR or real-time quantitative PCR.
Compared with the prior art, the invention has the following beneficial effects:
the PCR detection method can effectively improve the sample detection rate, ensure the stability and accuracy of the PCR detection result, reduce the inhibition of internal standards caused by different reasons in the experimental process, and reduce the laboratory sheet withdrawal rate.
Detailed Description
The present invention will be further described with reference to examples below in order to further explain the technical means adopted by the present invention and the effects thereof. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
Human papillomavirus HPV-DNA samples were tested.
Human papillomavirus HPV-DNA was characterized by 50 cases, 18 of which were positive samples.
Main reagents and instruments:
human papillomavirus HPV-DNA qualitative use of human papillomavirus nucleic acid typing detection kit (Shanghai perspective diagnosis technologies Co., ltd.);
a Bo-Ri amplifier;
luminex200 flow dot matrix instrument.
HPV-DNA qualitative sample testing procedures included:
step one, DNA extraction
Step two, PCR amplification
Step three, hybridization detection
And fourthly, detecting on-machine.
Method one
Step one, DNA extraction:
the preservation solution in the sample was transferred to the labeled EP tube, the supernatant was discarded after centrifugation of the EP tube, and then 200. Mu.L of a nucleic acid extraction reagent was added to the EP tube, and the plate was heated for 20 min and centrifuged for 5 min.
Step two, PCR amplification:
preparing a PCR reaction system:
taking out the PCR related reagent from the refrigerator, and standing at room temperature for 30 min to completely melt the frozen reagent; preparing a corresponding number of PCR reaction tubes according to the number of samples, for example, the number of samples is 16, 18 PCR reaction tubes are needed to be prepared by adding a positive quality control product and a negative quality control product, and marking the samples sequentially;
calculating the dosage of the premix, the primer mixed solution and the polymerase according to the number of samples detected in the experiment, adopting a pipetting gun to gently mix the premix, the primer mixed solution and the polymerase uniformly, and then sub-packaging the mixture into each PCR reaction tube according to 15 mu L/tube;
samples were added one by one to the corresponding PCR reaction tube in an amount of 5. Mu.L/tube, and after the cap was closed, the reaction liquid was concentrated at the bottom of the PCR reaction tube by centrifugation at 2000 rpm for 10 s. And (5) performing PCR amplification on the sample.
Amplification conditions: 95 ℃ for 5 min 1 cycle 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 5s 5 cycles 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 35s cycle 72 ℃ for 3 min 1 cycle, 4 ℃ for 10 min, and 4 ℃ for sealing and preserving for standby
Step three, hybridization detection:
according to the number of PCR tubes, the corresponding size of microwell hybridization plates were cut with scissors. Mixing microsphere hybridization solution by shaking, and adding 22 μL into hybridization hole; sucking 3 mu L of the PCR amplification product obtained in the second step, sequentially adding the PCR amplification product into the hybridization holes, and uniformly sucking and mixing; hybridization was performed by placing the hybridization plate in a metallic constant temperature bath.
Hybridization conditions: denaturation at 95℃for 5 min, hybridization at 48℃for 30 min and hybridization at 48℃for 15 min
After hybridization, 75. Mu.L of streptavidin-phycoerythrin was added rapidly and incubation was continued for 15 min at 48 ℃.
Fourth, on-machine detection:
and transferring the microporous hybridization plate to a preheated multifunctional flow dot matrix instrument for detection.
Interpretation of the results: when the contrast Globin signal in the sample is larger than 150 and the HPV type specific probe signal is larger than 150, judging that the HPV type corresponding to the probe is positive. If the control Globin signal in the sample is less than 150, an internal standard abnormality is indicated.
The results show that 50 samples of human papillomavirus HPV-DNA are qualitatively treated, the internal control Globin signals are less than 150, the internal standard is abnormal, and the results cannot be interpreted.
Method II
Step one, DNA extraction:
shaking the mixed sample at 23 ℃ to dissolve the cervical exfoliated cells on the cervical brush in preservation solution as much as possible, transferring the 0.4 mL sample into a 2 mL centrifuge tube, centrifuging for 3 min at 12000 r/min, discarding supernatant to leave a precipitate, respectively adding the washing agent 1 mL in table 1 along the EP tube wall, shaking 30s, mixing the precipitate, standing for 2 min at 37 ℃ in a metal bath, centrifuging for 3 min at 12000 r/min, discarding supernatant to leave a precipitate; adding the detergents 0.5 and mL in Table 1 along the EP pipe wall respectively, shaking for 30s, mixing, precipitating, standing in a metal bath at 37deg.C for 2 min, centrifuging for 3 min at 12000 r/min, and discarding supernatant to obtain precipitate; the plate is heated for 20 min and centrifuged for 5 min.
Step two, PCR amplification and the same method;
step three, hybridization detection and the same method as the step one;
and fourthly, detecting on-machine, and the same method is adopted.
The results are shown in Table 1.
TABLE 1
Results: from the tabular data, it can be seen that the use of the Nacl solution+alcohol+KCl or MgCl of the present invention 2 Pretreatment method for buffer PCR detection experiment processes HPV-DNA sample to be detectedThe standard number can reach 50 cases, and the positive rate can reach 36%; after the sample to be detected of HPV-DNA is treated by using a Nacl solution and alcohol, the normal number of internal standards can reach 36 cases, and the positive rate can reach 28%; the method is not in the range of the mass percent of the ethanol defined by the invention, the normal number of the internal standard is only 33, and the positive rate is 24%; after 80% NaCl solution is adopted to treat HPV-DNA samples to be detected, the normal number of internal standards can reach 21 cases, and the positive rate can reach 20%; after HPV-DNA samples to be detected are treated by using 90% NaCl solution, the normal number of internal standards can reach 21 cases, and the positive rate can reach 22%; and is not in the range of the mass percent of the Nacl solution defined by the invention, the normal number of the internal standard is only 18, and the positive rate is 18%.
Example 2
And detecting the GBS-DNA sample of the group B streptococcus.
Group B Streptococcus GBS-DNA qualitative 15 cases, total positive samples 2 cases.
Main reagents and instruments:
group B streptococcus GBS-DNA qualitative use of Group B Streptococcus (GBS) nucleic acid detection kit (PCR-fluorescent probe method) (Beijing Boer Chengcheng technology Co., ltd.)
ABI7500 real-time fluorescence PCR instrument.
Method one
Step one, DNA extraction:
sterile saline 1 mL was added to the sample, mixed well and transferred to an EP tube, centrifuged at 13000 rpm for 5 min, and the supernatant discarded. Adding 50 μLDNA extract into the precipitate, mixing, heating at 100deg.C for 10 min, and centrifuging at 13000 rpm for 10 min.
Step two, preparing a reaction system
The PCR reaction was removed, thawed at room temperature and centrifuged at 2000 r/min for 10 s. Subpackaging the sample into 8 connecting tubes according to the number of the sample, subpackaging 35 mu L of the sample into each hole, taking 5 mu L of sample supernatant, sequentially adding the sample supernatant into a PCR reaction liquid tube, and finally, obtaining a volume of 40 mu L; then centrifuging for 10s at 2000 r/min to avoid hanging beads on the tube;
step three, PCR amplification
Amplification conditions: UNG reaction: pre-denaturation at 50 ℃ for 20 min: 95℃for 5 min, PCR: FAM and HEX channel fluorescence signals were detected at 95℃for 15s,60℃for 35s,45 cycles, and 60℃respectively.
Interpretation of the results: GBS-DNA qualitative results amplification curves were normally in the form of standard S-shapes. If the FAM channel has no ct value, and the HEX channel signal is normal, the FAM channel is negative. If the FAM channel ct of the sample is less than or equal to 38, the sample is positive. If the HEX channel has no signal, the internal standard is abnormal.
The results show that 15 samples of group B streptococcus GBS-DNA are characterized in that HEX channels are all without signals, internal standards are abnormal, and the results cannot be interpreted.
Method II
Step one, DNA extraction:
adding sterile physiological saline 1 mL of 23 ℃ into a sample, uniformly mixing, transferring to a 1.5mL EP tube, centrifuging 12000 r/min for 8min, discarding supernatant precipitate, respectively adding the detergents 1 mL in table 2 along the EP tube wall, vibrating 30s, uniformly mixing the precipitate, standing in a metal bath at 37 ℃ for 2 min, centrifuging 12000 r/min for 5 min, discarding supernatant precipitate, respectively adding the detergents 0.5 mL in table 2 along the EP tube wall again, vibrating 30s, uniformly mixing the precipitate, standing in a metal bath at 37 ℃ for 2 min, centrifuging 12000 r/min for 5 min, discarding supernatant precipitate; adding 50 μl of nucleic acid extraction reagent, shaking, mixing well for 30s, heating at 100deg.C in metal bath for 10 min to allow cell lysis, centrifuging for 8min at 12 r/min, and collecting supernatant.
Step two, PCR amplification and the same method one.
The results are shown in Table 2.
TABLE 2
Results: from the tabular data, it can be seen that the use of the Nacl solution+alcohol+KCl or MgCl of the present invention 2 The normal number of internal standards after the pretreatment method of the buffer PCR detection experiment processes the group B streptococcus GBS-DNA qualitative sample can reach 15 cases, and the positive rate can reach 13.3%; after the qualitative sample of the GBS-DNA of the group B streptococcus is treated by using a NaCl solution and alcohol, the normal number of internal standards can reach 11 cases, and the positive rate can reach 6.67%; by mass percentage of ethanol not defined in the inventionIn the range, the normal number of the internal standard results is only 8, and the positive rate is 0%; after the group B streptococcus GBS-DNA qualitative sample is treated by adopting 80% NaCl solution, the normal number of internal standard can reach 8 cases, and the positive rate can reach 0%; after the group B streptococcus GBS-DNA qualitative sample is treated by adopting 90% NaCl solution, the normal number of internal standard can reach 8 cases, and the positive rate can reach 0%; and is not in the range of the mass percent of the Nacl solution defined by the invention, the normal number of the internal standard is only 7, and the positive rate is 0%.
Example 3
Three qualitative samples for detecting venereal disease.
Three qualitative cases of venereal disease are 15, wherein the total number of positive samples is 1.
Main reagents and instruments:
the three qualitative applications of venereal disease are ureaplasma urealyticum, chlamydia trachomatis, gonococcus nucleic acid detection kit (PCR-fluorescent probe method) (Sanxiang Biotech Co., ltd.)
ABI7500 real-time fluorescence PCR instrument
Method one
Step one, DNA extraction:
adding 1 mL sterile physiological saline into the sample collecting tube, fully vibrating and uniformly mixing, and then pouring all the liquid into a 1.5mL sterile centrifuge tube to serve as a sample to be tested.
Preparing a reaction system:
the PCR reagent (reaction solution 38. Mu.L/human+enzyme mixture 2. Mu.L/human+internal standard 1. Mu.L/human) was taken out, thawed at room temperature and centrifuged at 2000 r/min for 10 s. Subpackaging the sample into 8 connecting tubes according to the number of the sample, subpackaging 40 mu L of the sample supernatant into 10 mu L of each hole, sequentially adding the sample supernatant into a PCR reaction liquid tube, and finally obtaining a volume of 50 mu L; centrifuging for 10s at 2 r/min to avoid hanging beads on the tube;
step three, PCR amplification:
amplification conditions: UNG enzyme reaction at 50deg.C for 2 min, taq enzyme activation at 94deg.C for 5 min, denaturation at 94deg.C for 15s, annealing, extension at 57 deg.C for 30s for 45 cycles, detection of FAM and HEX channel fluorescence signals at 57 deg.C, respectively.
Qualitative results of three items (UU-DNA, CT-DNA, NG-DNA) were determined: if the measured ct value is less than or equal to 38, the amplification curve is an obvious S-shaped sample, and the sample is positive; if the internal standard ct value is more than 40 or is not displayed, the internal standard is abnormal.
The results show that in three qualitative 15 samples of venereal diseases, the internal standard ct values of 6 samples are larger than the internal standard ct values of 40,9 samples, the internal standard is abnormal, and the results cannot be interpreted.
Method II
Step one, DNA extraction:
adding sterile physiological saline 1 mL of the sample, uniformly mixing at the temperature of 23 ℃, transferring to a 1.5mL EP tube, centrifuging at 12000 r/min for 8min, discarding supernatant to leave a precipitate, respectively adding the detergents 1 mL in the table 3 along the EP tube wall, vibrating for 30s, uniformly mixing the precipitate, standing in a metal bath at 37 ℃ for 2 min, centrifuging at 12000 r/min for 5 min, discarding supernatant to leave a precipitate, and performing sedimentation; adding the detergents 0.5 and mL in Table 3 along the EP pipe wall respectively, shaking for 30s, mixing, precipitating, standing in a metal bath at 37deg.C for 2 min, centrifuging for 5 min 12000 r/min, and discarding supernatant to obtain precipitate; adding 50 μl of release agent, shaking, mixing for 30s, standing at 23deg.C for 10 min to allow cells to be completely lysed, centrifuging for 10s at 12 r/min, and collecting supernatant.
And step two, preparing a reaction system, and adopting the same method.
And thirdly, PCR amplification, which is the same as the first method.
The results are shown in Table 3.
TABLE 3 Table 3
Results: from the tabular data, it can be seen that the use of the Nacl solution+alcohol+KCl or MgCl of the present invention 2 The normal number of internal standards after three qualitative samples of venereal diseases are treated by a pretreatment method of a buffer PCR detection experiment can reach 15 cases, and the positive rate can reach 6.67%; after three qualitative samples of venereal disease are treated by using a Nacl solution and alcohol, the normal number of internal standards can reach 11 cases, and the positive rate can reach 0%; the method is not in the range of the mass percent of the ethanol defined by the invention, the normal number of the internal standard results is only 9, and the positive rate is 0%; treatment of three qualitative samples of venereal disease with 80% NaCl solutionThe normal number of the internal standard can reach 9 cases, and the positive rate can reach 0%; after three qualitative samples of venereal disease are treated by using 90% NaCl solution, the normal number of internal standard can reach 90 cases, and the positive rate can reach 0%; and is not in the range of the mass percent of the Nacl solution defined by the invention, the normal number of the internal standard is only 8, and the positive rate is 0%.
In conclusion, the PCR method can effectively improve the detection rate of positive samples, ensure the stability and accuracy of the PCR detection result, reduce the abnormal conditions of internal standards caused by different reasons in the experimental process, and reduce the single withdrawal rate in a laboratory.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (7)

1. A method of PCR detection, the method comprising: treating a sample to be detected by using a washing solution to extract DNA, heating and centrifuging to obtain target DNA, hybridizing an amplified product with microspheres after PCR amplifying the target DNA, and carrying out fluorescence value detection on hybridization; the washing liquid comprises a combination of a Nacl solution and ethanol, wherein the Nacl solution comprises 80-90% by mass of Nacl; the mass percentage of the ethanol is 70-75%.
2. The method of claim 1, wherein the sample to be tested comprises any one or a combination of at least two of human papillomavirus HPV-DNA, group B streptococcus qualitative GBS-DNA, or a qualitative sample of a venereal disease.
3. The method of claim 1, wherein the wash liquor further comprises isopropyl alcohol; the volume ratio of the ethanol to the isopropanol in the washing liquid is (0.5-3): 1.
4. The method of claim 3, wherein the wash solution further comprises a buffer.
5. The method of claim 4, wherein the buffer comprises KCl solution or MgCl 2 A solution; the pH of the buffer is 4-6.
6. The method according to claim 3, wherein the volume ratio of the NaCl solution, the alcohol and the buffer in the washing liquid is 1 (1-3): 1.
7. Use of the method of any one of claims 1-6 in SSR-PCR, ISSR-PCR, RACE-PCR, touchdown PCR or real-time quantitative PCR.
CN202410077552.6A 2024-01-19 2024-01-19 PCR detection method and application thereof Pending CN117587109A (en)

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