CN112195237A

CN112195237A - Digital PCR detection kit for detecting fetal free DNA content and method thereof

Info

Publication number: CN112195237A
Application number: CN202011291044.6A
Authority: CN
Inventors: 祝令香; 王芳; 陆超; 杨文军
Original assignee: Beijing Targeting One Biotechnology Co ltd; Institute Of Science And Technology National Health Commission
Current assignee: Beijing Targeting One Biotechnology Co ltd; Institute Of Science And Technology National Health Commission
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2021-01-08

Abstract

The invention provides a digital PCR detection kit for detecting the content of free DNA of a fetus and a method thereof, wherein the kit comprises digital PCR detection reagents which are respectively used for detecting a CpG island-containing region of a fetal marker gene, a methylation-sensitive restriction enzyme digestion site-containing region of an internal reference gene and a methylation-sensitive restriction enzyme digestion site-free region of the internal reference gene. The kit and the method thereof have accurate quantification and no sex selectivity, and are a universal reagent and a method for detecting fetal free DNA in maternal plasma.

Description

Digital PCR detection kit for detecting fetal free DNA content and method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a digital PCR detection kit for detecting the content of free DNA of a fetus and a method thereof.

Background

The non-invasive prenatal DNA detection technology (NIPT) is widely applied in clinic, and researches show that the peripheral blood of pregnant women contains fetal free DNA (cffDNA), and the prenatal diagnosis and screening of the birth defects can be realized by detecting the fetal free DNA. Screening and detection of fetal chromosomal aneuploidies has been currently accomplished by next generation sequencing technologies (NGS). However, when the technology is used, false negative can occur due to the fact that the cffDNA concentration is too low, and therefore, the cffDNA concentration higher than 4% is considered to be a very important quality control index; in addition, in noninvasive monogenic disease detection, accurate estimation of cffDNA concentration is more important, and the cffDNA concentration is an important parameter for analyzing site variation.

cffDNA is derived from placental apoptotic trophoblasts and is shorter than maternal free DNA, approximately 140-145 bases in length, whereas maternal cfDNA is typically approximately 166bp in length. cffDNA represents on average 10-20% of the total maternal plasma cfDNA, which varies from individual to individual and generally increases with gestational age. After delivery, cffDNA is cleared from maternal plasma within hours.

The current method for detecting the proportion of fetal free DNA depends mainly on the Y chromosome sequence (such as SRY, DYS14 gene), but the method is only effective for male fetus. It was found that maternal and fetal free DNA differ in methylation appearance modification, and therefore techniques for detection based on Differential Methylation Region (DMR) were developed, including bisufite treatment, where cytosines without methylation modification are converted to uracils by bisufite treatment, and the percentage of methylation modification at each cytosine site can be subsequently obtained by PCR-sequencing or second-generation sequencing techniques. The method is accurate as a gold standard for methylation detection, but the conversion efficiency of the bisulfate treatment is very important, and the conversion efficiency of the currently commercialized bisulfate kit can reach more than 99%. The problem of bisufite treatment is that the steps are complicated, the loss of DNA after treatment is high, so the amount of DNA required is high, the current commercial kit generally needs more than 20ng of DNA for treatment, in addition, the DNA breakage damage after bisufite treatment is serious, and for free DNA, high sensitivity is difficult to achieve.

Another method is restriction enzyme treatment (hereinafter referred to as enzyme cutting method for short), which is to digest a DNA sample with methylation-sensitive restriction enzyme, design a primer for a specific enzyme cutting site, and detect an enzyme cutting product by PCR (polymerase chain reaction) so as to judge whether the site has methylation modification and the modification ratio. In addition, the method generally has long treatment time at present, and different restriction enzymes are required to be added step by step so as to achieve the purpose of complete digestion.

Digital PCR is a technique suitable for quantifying nucleic acid, and the technique generates droplets of several micrometers to hundreds of micrometers by means of a microfluidic chip, and the droplets can be regarded as test tubes of a pico-liter/nano-liter scale to wrap single molecules for PCR reaction. The unique advantages of digital PCR are as follows: (1) the sensitivity is high, one liquid drop can contain single molecules or cells, and single-molecule-level detection is realized at a physical layer; (2) the accuracy is strong, millions of micro-droplets can be generated by the micro-fluidic chip each time, the micro-droplets are counted one by one, the number of the templates is calculated through Poisson distribution, digitization can be realized, and the conclusion is reliable.

The invention relates to a kit and a method for quantitatively detecting the content of free DNA of a fetus based on an enzyme digestion method and a digital PCR (polymerase chain reaction), which are optimized and improved aiming at the defects of long reaction time, most overnight digestion (16 hours), incomplete reaction and the like of the enzyme digestion method.

Disclosure of Invention

In order to solve the above problems, the present invention provides a digital PCR detection kit for detecting fetal free DNA content, the kit comprises digital PCR detection reagents for detecting a CpG island-containing region of a fetal marker gene, a methylation-sensitive restriction enzyme digestion site-containing region of an internal reference gene, and a methylation-sensitive restriction enzyme digestion site-free region of the internal reference gene, respectively, wherein the CpG island-containing region of the fetal marker gene is a region of differential methylation expression between a fetus and a mother, and can be distinguished by the methylation-sensitive restriction enzyme, and a sequence of the region in the mother is removed, thereby obtaining the fetal free DNA content; and the digital PCR detection reagent comprises a first detection probe, a second detection probe and a third detection probe which are respectively used for detecting a CpG island region of a fetal marker gene, a region of an internal reference gene containing a methylation-sensitive restriction enzyme digestion site and a region of the internal reference gene not containing the methylation-sensitive restriction enzyme digestion site, wherein the first detection probe is marked by a first fluorescence, the second detection probe is marked by the first fluorescence and a second fluorescence respectively, and the third detection probe is marked by the second fluorescence. In the invention, 3 detection targets can be distinguished according to the positions of signal groups by using two fluorescent labels, wherein the first detection probe and the third detection probe are respectively arranged in two vertical directions with a full negative liquid drop as an origin, the second detection probe simultaneously comprises the first fluorescent label and the second fluorescent label, and the two fluorescent labels generate signals in the micro-liquid drop, so that the second detection probe can be positioned above the origin to be distinguished from the positions of the first probe and the third probe.

In one embodiment, the digital PCR detection kit comprises a methylation sensitive restriction enzyme reagent that is a combination of multiple restriction enzymes.

In one embodiment, the restriction enzyme cleavage site is GCGC or CCGG.

In one embodiment, the restriction enzyme reagent is a combination of a BstUI restriction enzyme, a HhaII restriction enzyme, and a BstY1 restriction enzyme.

In one embodiment, the fetal marker gene is RASSF1A gene, and/or the reference gene is ACTB gene.

In one embodiment, the upstream primer for digital PCR amplification of RASSF1A gene is SEQ ID NO: AGCCTGAGCTCATTGAGCTG, the downstream primer is SEQ ID NO: AGCCTGAGCTCATTGAGCTG, and the probe for detecting the RASSF1A gene is SEQ ID NO: CCAACGCGCTGCGCA, 3: CCAACGCGCTGCGCA.

In one embodiment, the upstream primer for digital PCR amplification of the ACTB gene containing a methylation sensitive restriction endonuclease cut site region is SEQ ID NO: AGCCTGAGCTCATTGAGCTG, the downstream primer is SEQ ID NO: CGGCGGATCGGCAAA, and the detection probe is SEQ ID NO: ACCGCCGAGACCGCGTC, 6: ACCGCCGAGACCGCGTC.

In one embodiment, the upstream primer for digital PCR amplification of the ACTB gene that does not contain a methylation sensitive restriction endonuclease cut site region is SEQ ID NO: GCATCCCCCAAAGTTCAC, the downstream primer is SEQ ID NO: CGCATCTCATATTTGGAATGAC, and the detection probe is SEQ ID NO: GTGGCCGAGGACTTTGATTGC in a ratio of 9: GTGGCCGAGGACTTTGATTGC.

In one embodiment, the invention provides a digital PCR detection method for detecting fetal free DNA content, the method comprises detecting a CpG island-containing region of a fetal marker gene, a methylation-sensitive restriction enzyme cleavage site-containing region of an internal reference gene, and detecting a methylation-sensitive restriction enzyme cleavage site-free region of the internal reference gene, wherein the CpG island-containing region of the fetal marker gene is a region of differential methylation expression between a fetus and a mother, and can be distinguished by methylation-sensitive restriction enzyme to remove a sequence of the region in the mother, thereby obtaining the fetal free DNA content; and the digital PCR reagent comprises a CpG island region, an internal reference gene restriction enzyme cutting site region and an internal reference gene restriction enzyme cutting site region which are respectively detected by a first detection probe, a second detection probe and a third detection probe, wherein the first detection probe is marked by a first fluorescence, the second detection probe is marked by a first fluorescence and a second fluorescence respectively, and the third detection probe is marked by a second fluorescence.

The digital PCR detection kit for detecting the content of the free DNA of the fetus combines the digital PCR technology and the restriction endonuclease method, and realizes the quantitative detection of the content of the free DNA of the fetus. When the digital PCR detection kit for detecting the content of free DNA of the fetus is used for detection, the reaction time is greatly shortened compared with that of a common restriction endonuclease method, all reaction raw materials are added into the kit at one time, the operation is greatly simplified, and because the digital PCR redistributes the reaction system when liquid drops are generated, the subsequent enzyme adding operation step is not carried out any more. Therefore, the present invention can complete the digestion of restriction enzyme and make the subsequent digital PCR compatible. The invention can quantify the methylation of the fetal marker gene, thereby quantifying the content of the fetal free DNA in the maternal body, the total amount of the maternal free DNA is quantified through an internal reference gene segment without enzyme cutting sites, the restriction enzyme digestion is the premise of accurate quantification, otherwise, the quantitative result of the fetal free DNA is higher, therefore, the invention further controls the enzyme cutting effect through the detection result of the internal reference gene segment containing the enzyme cutting sites, when the segment is not detected, the enzyme cutting is complete, and the result is compared with the quantitative result of the male fetus specific gene SRY by the method, which shows that the invention has accurate quantification and no sex selectivity, and is a universal detection mode of the fetal free DNA in the maternal plasma.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic view of the detection principle of the kit of the present invention;

FIG. 2 is a graph showing the correlation between RASSF1A methylase cleavage and SRY quantitative fetal free DNA (cfDNA) ratio.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. The invention is further described with reference to the following figures and examples.

Embodiment one invention detection principle

The detection principle of the invention is shown in figure 1: primer probes are respectively designed aiming at the CpG island region of RASSF1A and the region containing the enzyme cutting site of the ACTB gene, and are used as the action target of methylation sensitive restriction enzyme, and meanwhile, the region not containing the methylation sensitive restriction enzyme cutting site of the ACTB gene is used as the amplification target and is used for quantifying the total amount of DNA. Free DNA was digested with multiple methylation-sensitive restriction enzymes, and the ratio of undigested methylated RASSF1A (representing fetal cffDNA content) to total free cfDNA (ACTB content without restriction sites) was quantitatively determined by digital PCR as the proportion of free cffDNA. The specific description is as follows:

(1) the RASSF1A gene amplification target region is designed in CpG island region, which is the region of differential methylation expression of fetus and mother, and can be distinguished by methylation sensitive restriction endonuclease, and the sequence of the region in the mother can be removed, thereby obtaining the content of fetus free DNA.

(2) Two target regions were designed for the ACTB gene, which is hypomethylated both maternal and fetal: a region (ACTB-1 in the figure) containing a plurality of enzyme cutting sites for controlling whether the enzyme cutting effect is complete; the other is a region not containing a cleavage site (ACTB-2 in the figure) for quantifying the total amount of free DNA.

In fig. 1, a: cfDNA is not digested, and all targets have signals; b: after the cfDNA of the pregnant woman is cut by enzyme, ACTB-1 (containing a plurality of enzyme cutting sites) is completely cut by enzyme without signals, RASSF1A fetal cffDNA is methylated and not cut by enzyme with signals, and ACTB-2 has no enzyme cutting sites and is not cut by enzyme with signals; c: the non-pregnant woman cfDNA is subjected to enzyme digestion, ACTB-1 (containing a plurality of enzyme digestion sites) is completely digested without signals, RASSF1A is not methylated and completely digested without signals, and ACTB-2 has no enzyme digestion sites, is not digested with signals and has signals.

EXAMPLE two kits of the invention and uses thereof

1. Extraction of cfDNA

DNA extraction was carried out with 1-2ml of plasma using free DNA extraction kit (Tiangen organisms) according to the instructions, and the final DNA eluate was stored at-20 ℃ for further use.

2. Digital PCR amplification system

30 mu L of amplification system, 1 in final concentration

Buffer (NEB), 1 × supermix (New Youji biology), primers and probes containing RASSF1A, ACTB-1 (containing enzyme cutting sites) and ACTB-2 (without enzyme cutting sites) in the detection system, wherein each primer is 600nM, and each probe is 300 nM; primer probe sequences are shown in Table 1.

The digestion conditions were set according to the experiment, and the PCR cycling conditions were as follows: 95 ℃ for 10min, 95 ℃ for 30 seconds and 60 ℃ for 60 seconds, 45 cycles.

TABLE 1 primer Probe sequences

3. Methylation sensitive enzyme combinatorial screening and optimization assays

After cfDNA extracted from a non-pregnant plasma sample is mixed, enzyme combination digestion screening and reaction time optimization are carried out, an amplification reaction system is as above, the used cfDNA is 15 mu L, different enzyme combinations are added, different reaction times are carried out (shown in table 2), and then enzyme digestion reaction and digital PCR reaction are carried out on a PCR thermal cycler. Samples without enzyme were used as a control to determine whether cfDNA was completely digested. Because non-pregnant woman cfDNA was used, complete digestion was considered if no amplification of RASSF1A sequence and ACTB-1 sequence was detected. The detection results are shown in table 2, the methylation sensitive BstUI enzyme alone can not achieve complete digestion after 3 hours of action, so that digestion is performed by adopting a combination of a plurality of enzymes, and it can be seen that the combination of BstUI + HhaII + BstY1 has better effect than the former two enzymes, and the BstYI is selected so that the enzyme does not basically contain the enzyme cleavage site in the detection regions of the two genes, but the enzyme can digest a larger fragment in free DNA, thereby improving the cleavage efficiency of the other two enzymes, and simultaneously for digital PCR, the complexity of the template is reduced, so that the template is easily dispersed into different droplets, thereby improving the detection sensitivity. When BstUI was used in an amount of 50U, the unmethylated enzyme-containing cleavage site region was essentially completely digested, no copies of RASSF1A and ACTB-1 were detected, and ACTB-2 was normally detected. The enzyme digestion reaction is combined with the subsequent digital PCR reaction, and the enzyme digestion reaction is used as a restriction enzyme inactivation program at 95 ℃ for 10 minutes without influencing the result detection.

TABLE 2 digestion test with different enzyme combinations

4. Digestion time shortening test

The main problem of the enzyme cutting method is longer reaction time, so the invention further examines whether shortening the digestion time is feasible, uses non-pregnant plasma cfDNA as a template, uses 50U BstUI +50U HhaII +10U BstYI for combined digestion, the digestion time is shown in Table 3, when the digestion time is 30min, a better complete digestion effect can be achieved, and when the digestion time is 15min, a trace amount of undigested fragments still exist. Thus, the digestion time was determined to be 30 min.

TABLE 3 different digestion time tests

5. Test for shortening inactivation time

After digestion with restriction endonuclease, inactivation treatment is needed, otherwise the subsequent digital PCR reaction may be affected, because the PCR product synthesized subsequently is not protected by methylation and may be degraded by the restriction endonuclease, but in the digital PCR, because the template is already segmented at the time of initial droplet generation, if the restriction endonuclease is not completely inactivated, the PCR product amount in the positive droplet is mainly affected, thereby affecting the signal intensity, and theoretically not affecting the quantitative result. After mixing cfDNA extracted from a plasma sample of a pregnant woman, optimizing enzyme inactivation time, carrying out combined digestion by using 50U BstUI +50U HhaII +10U BstYI, carrying out enzyme digestion treatment for different times (shown in Table 4) by using an amplification reaction system as above and 15 mu L of cfDNA, and then carrying out enzyme digestion reaction and digital PCR reaction on a PCR thermal cycler. And (4) taking the sample without the enzyme as a control group, and determining whether the inactivation time influences the detection effect. Because cfDNA of pregnant women is used, theoretically a proportion of RASSF1A sequence should be detected, but ACTB-1 sequence is unmethylated and should be digested without amplification. The results are shown in Table 4, and different inactivation times did not affect the results. The quantified fetal cffDNA ratios were consistent.

TABLE 4 inactivation time test

Effect of DNA template input

Whether the restriction enzyme can completely digest the DNA template is also in a large relation with the template input amount, theoretically, the higher the DNA amount is, the longer the enzyme amount and the reaction time are, the non-pregnant plasma sample cfDNA is mixed, then the sample cfDNA is concentrated by 5 times by using a vacuum concentrator (Eppendorf), the DNA template input amount is verified, the digestion is carried out by using a combination of 50U BstUI +50U HhaII +10U BstYI, the digestion and inactivation time is 30min +95 ℃ for 10min, the amplification reaction system is as above, the used cfDNA amount is 3, 9, 18 and 45ng (as shown in Table 5), and then the enzyme digestion reaction and the digital PCR reaction are carried out on a PCR thermal cycler. It was determined whether complete digestion of DNA could be achieved at high DNA template input. As shown in Table 5, when the amount of the DNA template input reached 45ng (135000 copies in theory), complete digestion was still possible.

TABLE 5 DNA template input

Amount of DNA template	RASSF1A copy number	ACTB-1 copy number	ACTB-2 copy number
				3ng
	0	0	907.3
				9ng	0	1.2	2465.4
18ng	0	1.3	5221.8
				45ng	1.2	0	12276.2

7. Clinical sample testing

The test is carried out by 10 pregnant woman samples carrying male fetus, cfDNA is extracted, the combined digestion is carried out by using 50U BstUI +50U HhaII +10U BstYI, the digestion and inactivation time is 30min +95 ℃ for 10min at 37 ℃, the amplification reaction system is as above, and the comparison with SRY/ACTB-2 control reaction is carried out, so that the accuracy of the detection method is determined. The results are shown in Table 6, and the quantitative results of RASSF1A methylase cleavage and SRY are basically consistent, the correlation is high, and the correlation coefficient R2 is 0.9974, so that the method has accuracy and no sex selectivity, and can be used as a universal method for quantifying the proportion of free fetal DNA.

TABLE 6 test results of clinical specimens

It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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Claims

1. A digital PCR detection kit for detecting the content of fetal free DNA is characterized by comprising digital PCR detection reagents which are respectively used for detecting a CpG island-containing region of a fetal marker gene, a methylation-sensitive restriction enzyme digestion site-containing region of an internal reference gene and a methylation-sensitive restriction enzyme digestion site-free region of the internal reference gene, wherein the CpG island-containing region of the fetal marker gene is a region for differential methylation expression of a fetus and a mother body and can be distinguished by the methylation-sensitive restriction enzyme, and a sequence of the region in the mother body is removed, so that the content of the fetal free DNA is obtained; and

the digital PCR detection reagent comprises a first detection probe, a second detection probe and a third detection probe which are respectively used for detecting a CpG island region of a fetal marker gene, a region of an internal reference gene containing a methylation-sensitive restriction enzyme cutting site and a region of the internal reference gene not containing the methylation-sensitive restriction enzyme cutting site, wherein the first detection probe is marked by a first fluorescence, the second detection probe is marked by the first fluorescence and a second fluorescence respectively, and the third detection probe is marked by the second fluorescence.

2. The digital PCR detection kit of claim 1, wherein the digital PCR detection kit comprises a methylation sensitive restriction enzyme reagent, wherein the restriction enzyme reagent is a combination of a plurality of restriction enzymes.

3. The digital PCR detection kit according to claim 2, wherein the restriction enzyme cleavage site is GCGC or CCGG.

4. The digital PCR detection kit of claim 3, wherein the restriction enzyme reagent is a combination of BstUI restriction enzyme, HhaII restriction enzyme and BstY1 restriction enzyme.

5. The digital PCR assay kit of claim 1, wherein the fetal marker gene is RASSF1A gene and/or the reference gene is ACTB gene.

6. The digital PCR detection kit according to claim 5, wherein the upstream primer for amplifying RASSF1A gene by digital PCR is SEQ ID NO: AGCCTGAGCTCATTGAGCTG, the downstream primer is SEQ ID NO: AGCCTGAGCTCATTGAGCTG, and the probe for detecting the RASSF1A gene is SEQ ID NO: CCAACGCGCTGCGCA, 3: CCAACGCGCTGCGCA.

7. The digital PCR detection kit according to claim 5, wherein the upstream primer for amplifying the region of the ACTB gene containing the methylation sensitive restriction endonuclease cleavage site is SEQ ID NO: AGCCTGAGCTCATTGAGCTG, the downstream primer is SEQ ID NO: CGGCGGATCGGCAAA, and the detection probe is SEQ ID NO: ACCGCCGAGACCGCGTC, 6: ACCGCCGAGACCGCGTC.

8. The digital PCR detection kit according to claim 5, wherein the upstream primer for amplifying the region of the ACTB gene not containing the methylation sensitive restriction endonuclease site by digital PCR is SEQ ID NO: GCATCCCCCAAAGTTCAC, the downstream primer is SEQ ID NO: CGCATCTCATATTTGGAATGAC, and the detection probe is SEQ ID NO: GTGGCCGAGGACTTTGATTGC in a ratio of 9: GTGGCCGAGGACTTTGATTGC.

9. A digital PCR detection method for detecting the content of fetal free DNA is characterized by comprising the steps of detecting a CpG island-containing region of a fetal marker gene, a restriction enzyme cutting site region of an internal reference gene containing methylation sensitivity and detecting a restriction enzyme cutting site region of the internal reference gene not containing methylation sensitivity, wherein the CpG island-containing region of the fetal marker gene is a region with differential methylation expression between a fetus and a mother body, and can be distinguished by the methylation sensitivity restriction enzyme to remove a sequence of the region in the mother body, so that the content of fetal free DNA is obtained;

the method comprises the steps of detecting a CpG island region of a fetal marker gene, a region of an internal reference gene containing a methylation-sensitive restriction enzyme digestion site and a region of the internal reference gene not containing the methylation-sensitive restriction enzyme digestion site by using a first detection probe, a second detection probe and a third detection probe respectively, wherein the first detection probe is marked by a first fluorescence, the second detection probe is marked by the first fluorescence and a second fluorescence respectively, and the third detection probe is marked by the second fluorescence.