CN114622040A

CN114622040A - PCR primer group for detecting human cytomegalovirus in feces and micro-drop digital PCR kit thereof

Info

Publication number: CN114622040A
Application number: CN202210429231.9A
Authority: CN
Inventors: 陈丽婷; 周剑峰; 顾佳; 肖敏; 曹阳; 黄亮
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-06-14

Abstract

The invention discloses a PCR primer group for detecting human cytomegalovirus in feces and a micro-drop digital PCR kit thereof. The invention designs specific primers and MGB hydrolysis probes based on the gene sequence of the coding immediate early transcription regulatory protein in the human cytomegalovirus conserved AD169 genome, thereby obtaining the micro-drop digital PCR kit.

Description

PCR primer group for detecting human cytomegalovirus in feces and micro-drop digital PCR kit thereof

Technical Field

The invention relates to the field of virus nucleic acid detection, in particular to a PCR primer group for detecting human cytomegalovirus in feces and a micro-drop digital PCR kit thereof.

Background

The allogeneic hematopoietic stem cell transplantation is an important treatment mode for various blood system diseases, gastrointestinal symptoms often appear in transplanted patients, most possibly caused by acute graft-versus-host reaction and CMV enteritis, and are difficult to identify and diagnose as two common serious complications after the allogeneic hematopoietic stem cell transplantation, while the treatment modes clinically adopted aiming at the two different complications are quite different, so that the definite diagnosis and the adoption of active treatment measures are closely related to the success or failure of the allogeneic hematopoietic stem cell transplantation.

Currently, CMV enteritis diagnosis involves the following methods:

the gold standard for CMV enteritis diagnosis is intestinal mucosa biopsy pathological examination under an enteroscope, and the invasive operation aggravates the pain of patients with allogeneic hematopoietic stem cell transplantation, limits the material-taking position, cannot repeatedly sample for multiple times, cannot dynamically observe the change of virus copy number, possibly brings risks of intestinal bleeding and intestinal perforation, and greatly influences the treatment and prognosis of the patients.

2. Non-invasive methods, such as detection of CMV nucleic acids by fluorescent quantitative PCR reactions of stool samples of patients after xenotransplantation, are considered as potential alternatives to intestinal biopsies, and studies have shown that stool-based PCR detection is very valuable in the diagnosis of, or at least the exclusion of, CMV enteritis. There are also other studies that detect CMV in stool is an insufficiently qualified diagnostic modality for CMV enteritis.

Notably, DNA extraction procedures may have a critical impact on CMV detection in fecal samples.

1. First, stool specimens are a rather complex mixture in which components such as bile salts may interfere with many intermediate steps, including DNA purification and PCR reactions, by reducing the efficiency of DNA extraction and PCR amplification.

2. Second, DNA extraction procedures determine whether the major DNA type of the product is genomic DNA or episomal DNA, which is not fully considered for the differences in CMV DNA detection.

3. Finally, the conventional quantitative detection technique for nucleic acids is a fluorescent quantitative PCR detection, depends on a standard curve, has limited sensitivity, and is easy to generate non-specific fluorescent signals.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a PCR primer group for detecting human cytomegalovirus in excrement and a micro-drop digital PCR kit thereof; the invention designs specific primers and MGB hydrolysis probes based on the gene (IE1) sequence of the coding immediate early transcription regulatory protein in the conserved AD169 genome of the human cytomegalovirus, thereby obtaining the micro-drop digital PCR kit.

In order to achieve the aim, the invention designs a PCR primer group for detecting human cytomegalovirus in feces, the PCR primer group consists of a primer pair CMV-FR and a probe CMV-P1, wherein,

the primer pair CMV-FR is

Upstream primer CMV-F1:

5’-GTGATCCATGTGCTTATGACTTTGT-3’，

downstream primer CMV-R1: 5'-GCCTTGGTCACGGGTGTCT-3', respectively;

the probe CMV-P1: 5'-ATCATGTGTTTAGGCCC-3', and the 5 'end of the probe CMV-P1 is marked with a fluorescent group, and the 3' end is marked with a non-fluorescent quenching group.

Further, the fluorescent group is FAM, and the non-fluorescence quenching group is MGB.

The invention also provides a micro-drop digital PCR kit for detecting human cytomegalovirus in feces, wherein the PCR kit comprises the PCR primer group in the claim 1.

Further, the micro-drop digital PCR kit also comprises a digital PCR buffer solution, a micro-drop generating oil, a micro-drop generating card, a 96-well plate and an aluminum foil heat-sealing film.

Still further, the PCR kit also comprises an internal control gene primer group (designed according to the subunit gene POP4 of the coding human ribonucleoprotein complex), the internal control gene primer group comprises an internal control primer pair POP4-FR and an internal control probe POP4-P, wherein,

the internal control primer pair POP4 comprises

The upstream primer POP 4-F: 5'-GGCGGTGGTCCTGGAGTACT-3', downstream primer POP 4-R: 5'-AGAGGCCTTTGGCTTTCTTCTT-3', respectively;

the internal control probe primer POP 4-P: 5'-ACCCGCCACAAGC-3', and the 5 ' end of the internal control probe POP4-P is marked with a fluorescent group.

The invention also provides a method for detecting the human cytomegalovirus nucleic acid by using the micro-drop type digital PCR kit, which at least comprises the following steps:

1) treating the fecal specimen, and extracting free DNA of the fecal supernatant;

2) preparing a micro-drop digital PCR reaction mixed solution: mixing the micro-drop digital PCR detection reagent with a sample DNA template to be detected to obtain a micro-drop digital PCR reaction mixed solution;

3) droplet generation: adding the micro-drop digital PCR reaction mixed solution and micro-drop generating oil into a micro-drop generating card, and placing the micro-drop generating card in a micro-drop generating instrument to generate micro-drops;

4) and (3) PCR amplification reaction: transferring the generated microdroplets into a 96-well plate, covering an aluminum foil membrane, sealing the membrane by a membrane sealing machine, and putting the 96-well plate into a PCR (polymerase chain reaction) instrument for PCR amplification reaction after the membrane is sealed;

5) reading the fluorescence signal: and (3) placing the amplified 96-well plate in a microdroplet reader, setting parameters, and directly reading and analyzing results by using software.

6. The method of claim 5, wherein in step 3), the method for generating the microdroplets comprises the following steps:

adding the micro-drop digital PCR reaction mixed solution into a micro-drop generation plate, then adding micro-drop generation oil into the corresponding other holes, then placing the micro-drop generation plate into a micro-drop generation card, covering a leather sheath, and placing the micro-drop generation card into a micro-drop generator to perform micro-drop generation automatically.

Further, in the step 4), the system of the micro-drop digital PCR reaction mixture is as follows:

still further, in the step 4), the conditions of the PCR amplification reaction are: and (3) PCR amplification: keeping the temperature at 95 ℃ for 5min, then keeping the temperature at 95 ℃ for 30s, keeping the temperature at 60 ℃ for 1min, and carrying out 40 cycles (the temperature rise and fall speed is not more than 2.5 ℃/s); and finally, preserving the heat for 10min at the temperature of 98 ℃, and cooling to 4 ℃ for heat preservation.

The principle of the invention is as follows:

1. unlike genomic DNA, free DNA is widely present in various body fluids, such as serum, urine and fecal supernatant, and is easily collected. In recent years, there have been many dramatic advances in free DNA research, particularly in the fields of noninvasive early cancer diagnosis and prenatal screening, and free DNA sequencing has also been used to monitor infections in immunocompromised patients, with good agreement with clinical outcomes. The excrement free DNA is free DNA extracted after visible substances are removed from a dilute water sample, mainly comes from the DNA cracked, naturally apoptotic or secreted into an intestinal tract of an intestinal epithelial cell, after the intestinal epithelial cell is infected with pathogens, the patient and the pathogen DNA are released into the excrement due to the violent movement generated by the stimulation of the intestinal wall or the action of an immune system, and the excrement free DNA has rich genetic information and can become a better CMV nucleic acid detection specimen.

2. The micro-drop type digital PCR, as a third generation digital PCR technology, not only has higher sensitivity (10)^-4～10^-5) Can guide the diagnosis and treatment of pathogen infection with low copy number, and has better specificityGood guarantee, independent of standard curve, and absolute quantification. In the CMV assay, digital PCR also performed better than fluorescent quantitative PCR on stool samples that were easily inhibited.

The invention has the beneficial effects that:

the invention adopts the extraction method for extracting the free DNA of the excrement supernatant to replace the traditional excrement DNA extraction method, has higher DNA extraction yield and richer genetic information, can sample repeatedly, and realizes continuous monitoring. The kit adopted by the invention has the advantages of rapid detection, judgment of DNA extraction quality, strong specificity, high sensitivity, independence of a standard curve and realization of absolute quantification.

Drawings

FIG. 1 is a graph showing the effect of the method for extracting free DNA from fecal supernatant (method 1) compared with the conventional method for extracting DNA from feces (method 2);

FIG. 2 is a graph showing high amplification efficiency at 60 ℃ and negative and positive population clustering;

FIG. 3 is a graph showing the results of specific detection by the kit;

FIG. 4 is a graph showing the results of sensitivity testing of the kit;

FIG. 5 is a graph showing the results of the identity test of the kit;

FIG. 6 is a graph of the copy number of CMV and the corresponding clinical manifestations of a clinical case continuously monitored.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1

Extraction of free DNA from feces

1. Filtering a dilute water sample excrement specimen by using a 300-mesh filter membrane, transferring the sample into a 15ml centrifuge tube, centrifuging the sample for 5min by using a low-speed centrifuge at 3000rpm, transferring the supernatant into a new 15ml centrifuge tube, centrifuging the sample for 10min at 3000rpm, and removing excrement sediments as far as possible.

2. The extraction method of free DNA of the excrement supernatant comprises the following steps: free DNA Using the kit of Qiagen, Germany: (

Circulating Nucleic Acid Kit), the specific operation is as follows:

1) adding 2ml of centrifuged supernatant into a 50ml centrifuge tube, adding 1.6ml (1:0.8) of ACL lysate, 200 mu L of proteinase K (1:0.1) and 5 mu L of Carrier RNA (0.2 mu g/mu L), and covering the centrifuge tube with a cover to uniformly mix the mixture by shaking for 30 s;

2) placing a 50ml centrifuge tube on a centrifuge tube rack, placing the centrifuge tube in a water bath kettle at 60 ℃ for incubation for 30min, wherein the liquid level in the centrifuge tube needs to be lower than the water level of the water bath kettle so as to be fully heated;

3) adding 3.6ml (1:1.8) of ACB after the incubation is finished, covering a cover, shaking and mixing uniformly for 30s, and standing on ice for 5 min;

4) taking a centrifugal column, marking, adding a proper amount of 3) liquid (about 600. mu.L, not too much to avoid splashing when covering), centrifuging at high speed 12000rpm for 30s, discarding the receiving tube, replacing the receiving tube with a new one, and repeating the centrifugation step until all liquid is centrifuged. At the moment, the oven is opened and adjusted to 56 ℃;

5) adding 1600 mu L of ACW into the centrifugal column, centrifuging at high speed of 12000rpm for 1min, and replacing a new receiving tube;

6) adding 2750 μ L of washing solution ACW, centrifuging at high speed 12000rpm for 1min, and replacing with new receiving tube;

7) adding 750 μ L of absolute ethanol, centrifuging at high speed 14000rpm for 3min, replacing a new receiving tube, opening the cover of the column, placing the column in an oven, baking for 10min, preparing a new 1.5ml EP tube to collect cfDNA, labeling, placing the column in the EP tube, adding 25 μ L of AVE buffer, incubating for 3min, and centrifuging at 14000rpm for 1 min;

8) and (3) measuring the concentration: measuring the concentration of cfDNA of the feces supernatant by using a Qubit fluorometer 3.0 instead of measuring the concentration of the cfDNA by using a Nanodrop2000 method, wherein the concentration of the cfDNA is lower than 10 ng/. mu.L;

9) adjusting the concentration: if the concentration of cfDNA of the feces supernatant measured by the Nanodrop2000 exceeds 100 ng/mu L, the concentration needs to be diluted and adjusted within 100 ng/mu L.

Second, comparison of different extraction methods for paired samples

Collecting 22 diluted water sample specimens of patients with abdominal pain and diarrhea symptoms after allogeneic hematopoietic stem cell transplantation, equally dividing each specimen into two parts after the same treatment, and respectively carrying out a free DNA extraction method and a traditional excrement DNA extraction method, wherein the concentration of cfDNA of excrement supernatant is measured by using a Nanodrop2000, and the concentration is measured by using a Qubit fluorometer 3.0 instead of the concentration being lower than 10 ng/. mu.L.

As shown in fig. 1: it can be seen that the amount of free DNA obtained by the extraction method is larger than that obtained by the conventional extraction method (FIG. 1A). The paired DNA samples were subjected to digital PCR detection, and the copy number values of the internal control genes were compared (FIG. 1B), which shows that the quality of the nucleic acid samples was higher in the free DNA extraction method.

In addition, the amplification of internal control genes is not seen in three samples by using the traditional excrement DNA extraction method, but the extraction method of free DNA is obviously amplified. The method for extracting free DNA is more advantageous.

Example 2 design and screening of primer probes

1. Design of primers and probes:

the gene sequence of human cytomegalovirus gene IE1 is obtained by NCBI (national center for Biotechnology information) online tool, and specific primers and probes suitable for digital PCR are designed by using Primer Express 3.0.1 software, wherein alternative primers and probes are shown as follows:

F1：5’-GTGATCCATGTGCTTATGACTTTGT-3’

R1：5’-GCCTTGGTCACGGGTGTCT-3’

Probe1：5’-ATCATGTGTTTAGGCCC-3’

F2：5’-TGAAGCGCCGCATTGAG-3’

R2：5’-ATCGGCCCCCAGAATGTAC-3’

Probe2：5’-ATGAAGGTCTTTGCCC-3’

F3：5’-TGAAGCGCCGCATTGAG-3’

R3：5’-GGATCGGCCCCCAGAAT-3’

Probe3：5’-CATGAAGGTCTTTGCCCAGTA-3’

2. screening of probes and primers

1) Combining a primer and a probe: F1R1P1, F2R2P2, F3R3P3

2) Preparing a digital PCR reaction system: 10 μ L of 2x ddPCR Supermix, 2 μ L of each of the two sets of probes, 1 μ L of each of the two sets of upstream and downstream primers, 2 μ L of DNA sample, and 20 μ L of total volume. Generating a microdrop, amplifying, carrying out detection analysis by a microdrop analyzer, and finally determining the primer probe combination with the best specificity to be F1R1P 1; namely:

upstream primer CMV-F1:

5’-GTGATCCATGTGCTTATGACTTTGT-3’，

the downstream primer CMV-R1: 5'-GCCTTGGTCACGGGTGTCT-3', respectively;

probe CMV-P1: 5'-ATCATGTGTTTAGGCCC-3', and the 5 'end of the probe CMV-P1 is marked with FAM fluorescent group, and the 3' end is marked with MGB non-fluorescence quenching group.

Example 3

The kit is prepared by obtaining a primer group based on the screening, and the specific composition of the kit is shown in the following table:

the internal control gene primer group consists of an internal control primer pair POP4-FR and an internal control probe POP4-P, wherein,

the internal control primer pair POP4-FR comprises

internal control probe POP 4-P: 5'-ACCCGCCACAAGC-3', and the 5 ' end of the internal control probe POP4-P is marked with a fluorescent group.

Secondly, selecting the detection conditions of the kit and detecting the characteristics of the kit

1. Selection of annealing temperature

Preferred probe primer combinations are selected for droplet generation after formulation in a digital PCR system. The annealing temperature is set with 5 gradients of 57, 58, 59, 60 and 61 ℃, and a negative control is set at the same time, and digital PCR reaction is carried out. Analysis and comparison of the results show that the amplification efficiency is high and the negative and positive populations are clearly clustered under the condition of 60 ℃ (figure 2), so that 60 ℃ is selected as the annealing temperature in subsequent experiments.

2. Kit specificity detection

Using the above testThe kit detects 3 DNA specimens of healthy donors and 4 types of human herpesviruses (EB virus 1 x 10)³Copy number/. mu.L), human herpes virus type 6 (HHV-6B 1X 10)³Copy number/. mu.L) and human herpes virus type 5 (human cytomegalovirus 1X 10)³Copy number/. mu.L), 2 times of repetition, digital PCR amplification reaction, and observing whether specific reaction occurs.

After the DNAs of healthy donors, human herpesvirus type 4 (EB virus), human herpesvirus type 5 (human cytomegalovirus) and human herpesvirus type 6 (HHV-6B) were amplified by PCR, they were detected by a microdroplet reader, and the results were read and analyzed by QuantaSoft software.

As can be seen from FIG. 3, positive microdroplets were generated except for human herpesvirus type 5 (cytomegalovirus). The kit and the detection method have good specificity.

3. Kit sensitivity detection

Artificially synthesizing a micro-drop digital PCR amplified fragment (a sequence of a primer pair CMV-FR amplification), constructing a recombinant plasmid by taking a vector pUC57 as a vector, sequencing, and carrying out homology analysis on a sequencing result and a human cytomegalovirus gene sequence to ensure that the homology reaches 100%. Transforming into Escherichia coli competence DH5 alpha, shaking, extracting, performing HindIII and BamHI enzyme digestion on the large-quality-improved particles to linearize the large-quality-improved particles, recovering and purifying the linearized plasmid, measuring with a spectrophotometer, converting according to a formula, and diluting to 1 × 10⁴Copy number/. mu.L, a series of gradient standard products such as 1,2,3,4, 5,6,8,10,16,32,64 copy number/. mu.L are prepared, 6 duplicate wells are prepared for each concentration, digital PCR detection is carried out, nonlinear regression analysis is carried out according to the detection efficiency, and LOD50 (50% of detection limit) is obtained to be 2.844 copy number/. mu.L, and the result is shown in figure 4, which indicates that the kit has higher sensitivity.

4. Probe identity detection

By 1X 10 in the above-mentioned manner ⁴5 times gradient dilution is carried out on the copy number/mu L plasmid standard substance, the concentration gradients are 10000, 2000, 400, 80 and 16 respectively, each gradient is provided with three multiple holes and is subjected to digital PCR detection, the logarithm of 5 is respectively taken for linear analysis on the predicted copy number and the actual copy number, and FIG. 5 illustrates thatThe standard product results obtain a better linear relation, and stable detection results can be ensured.

EXAMPLE 4 clinical specimen testing

82 samples of 33 patients with abdominal pain and diarrhea after receiving allogeneic hematopoietic stem cell transplantation are collected, wherein the result of cytomegalovirus detection of 23 (28%) samples is positive, and the symptoms of the digestive tract of the patients are obviously relieved after the dosage of immunosuppressive drugs and the anti-human cytomegalovirus drugs are adopted. Taking a post-transplant patient who was monitored multiple times continuously as an example, the detected CMV gene copy number corresponded well with clinical performance, which better demonstrated that fecal supernatant free DNA detection could aid clinical diagnosis and guide treatment (fig. 6).

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

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Claims

1. A PCR primer group for detecting human cytomegalovirus in feces is characterized in that: the PCR primer group consists of a primer pair CMV-FR and a probe CMV-P1, wherein,

the primer pair CMV-FR is

Upstream primer CMV-F1:

5’-GTGATCCATGTGCTTATGACTTTGT-3’，

the downstream primer CMV-R1: 5'-GCCTTGGTCACGGGTGTCT-3', respectively;

2. The PCR primer set for detecting human cytomegalovirus in feces according to claim 1, wherein: the fluorescent group is FAM, and the non-fluorescence quenching group is MGB.

3. A microdroplet type digital PCR kit for detecting human cytomegalovirus in feces is characterized in that: the PCR kit comprises the PCR primer set of claim 1.

4. The digital PCR kit of claim 3, wherein: the micro-drop type digital PCR kit also comprises a digital PCR buffer solution, micro-drop generating oil, a micro-drop generating card, a 96-hole plate and an aluminum foil heat-sealing film.

5. The digital PCR kit of claim 3 or 4, wherein: the PCR kit also comprises an internal control gene primer group (designed according to the subunit gene POP4 of the coding human ribonucleoprotein complex), the internal control gene primer group comprises an internal control primer pair POP4-FR and an internal control probe POP4-P, wherein,

the internal control primer pair POP4 comprises

The upstream primer POP 4-F: 5'-GGCGGTGGTCCTGGAGTACT-3' the flow of the air in the air conditioner,

the downstream primer POP 4-R: 5'-AGAGGCCTTTGGCTTTCTTCTT-3', respectively;

the internal control probe POP 4-P: 5'-ACCCGCCACAAGC-3', and the 5 ' end of the internal control probe primer POP4-P is marked with a fluorescent group.

6. The method for detecting human cytomegalovirus nucleic acid by the microdroplet digital PCR kit of any one of claims 3 to 5, comprising at least the following steps:

4) PCR amplification reaction: transferring the generated microdroplets into a 96-well plate, covering an aluminum foil membrane, sealing the membrane by a membrane sealing machine, and putting the 96-well plate into a PCR (polymerase chain reaction) instrument for PCR amplification reaction after the membrane is sealed;

7. The method of claim 6, wherein in step 3), the method for generating the microdroplets comprises the following steps:

8. The method of claim 6, wherein in step 4), the system of the micro-drop digital PCR reaction mixture is as follows:

2x ddPCR Supermix 10μL upstream primer CMV-F1 1μL Downstream primer CMV-R1 1μL Probe CMV-P1 2μL Upstream primer POP4-F 1μL Downstream primer POP4-R 1μL Probe POP4-P 2μL DNA sample 2μL

9. The method of claim 6, wherein: in the step 4), the conditions of the PCR amplification reaction are as follows: and (3) PCR amplification: keeping the temperature at 95 ℃ for 5min, then keeping the temperature at 95 ℃ for 30s, keeping the temperature at 60 ℃ for 1min, and carrying out 40 cycles; and finally, preserving the heat for 10min at the temperature of 98 ℃, and cooling to 4 ℃ for heat preservation.