KR101387582B1 - Method for detecting cell free fetal nucleic acid in plasma of pregnant woman - Google Patents

Abstract

The present invention is directed to a method capable of effectively detecting cellular free fetal nucleic acid present in maternal blood. Prenatal screening due to low fertility and aging is preferred. So far, amniotic fluid testing has been dangerous and inaccurate, with half of mothers refusing to test. The present invention can detect fetal nucleic acid present in the blood safely and with high accuracy, and can be usefully used to identify and treat genetic analysis and disease of the fetus in a manner that is not dangerous to both mother and fetus.

Description

Method for detecting cell free fetal nucleic acid in plasma of pregnant woman}

The present invention is directed to a method capable of effectively detecting cellular free fetal nucleic acid present in maternal blood.

Early detection of pregnancy-related diseases, including the fetal genetic abnormalities, enables early medical intervention necessary for the safety of both the mother and the fetus. Amniotic fluid tests are being conducted to diagnose genetic abnormalities in these fetuses. Amniotic fluid surrounds the fetus and contains tissue cells of the fetus. Amniotic fluid test is a method of examining genetic abnormalities by taking DNA from tissue cells contained in the amniotic fluid. Amniocentesis is a routine test, but it is a conventional practice, as amniotic fluid leaks or infections, premature labor, premature labor, respiratory pain, fetal shock, fetal posture due to the bacterial infection of the amniotic membrane by the syringe and the wound through the syringe. There are risks of complications such as malformations and rhesus disease, and in serious cases, miscarriage. Therefore, there is an urgent need for a method that is not dangerous for both the mother and the fetus for genetic analysis and early diagnosis of the fetus.

On the other hand, 5 to 10% of the genetic material in the mother's blood (plasma and serum) is known to come from the fetus. In other words, in the blood of a pregnant woman, fragments of DNA or RNA flow through broken fetal placental cells (syncytiotrophoblast) due to apoptosis. Analyzing this fragmented DNA or RNA could lead to early diagnosis of fetal genetic disease. In particular, a method of detecting cell free fetal DNA (RNA) or cff DNA (RNA) in maternal blood has a merit of being safe as a non-invasive method, unlike amniocentesis, and research is being actively conducted. However, although fetal disease diagnosis can be effectively performed using the cellular free fetal nucleic acid present in the mother's blood, fetal nucleic acid present in a small amount and present in the state cleaved by nucleases present in the mother's blood. The relationship between the disease and the target gene is not actually confirmed due to the limitation of the method of confirming the presence or quantification thereof.

Under this background, the present inventors have made diligent efforts to devise a method to more effectively detect the cellular free fetal nucleic acid present in the mother in an extremely small amount, resulting in a continuous short length starting from the 5 'end of the fetal nucleic acid to be detected. When performing a polymerase chain reaction with a nucleic acid moiety having a target, it was confirmed that fetal nucleic acid in the mother can be effectively detected, and the present invention was completed.

An object of the present invention is to obtain a blood collected from the mother; From the 5 'end of the mRNA using a reverse transcription primer capable of complementarily binding to a portion of the 100th base sequence from the 5' end of the cell free fetal mRNA to be present in the blood Obtaining a cDNA consisting of a sequence complementary to RNA consisting of contiguous 20 to 100 base sequences; And it provides a method for detecting the nucleic acid of the fetus in the mother blood, comprising the step of identifying the amplification product obtained by performing a polymerase chain reaction with a primer set capable of amplifying the cDNA.

Another object of the present invention is to obtain a blood collected from the mother; And a primer set capable of complementarily binding to a part of the 100th base sequence from the 5 'end of the cell free fetal DNA to be present in the blood. It provides a method for detecting the nucleic acid of the fetus in the mother's blood, comprising the step of identifying an amplification product consisting of 20 to 100 consecutive sequences from the 5 'end of the DNA.

As one aspect for solving the above problems, the present invention provides a method for detecting the nucleic acid of the cell free fetal present in maternal blood. Specifically, the method of the present invention,

(a) obtaining blood collected from the mother;

(b) using a reverse transcription primer capable of complementarily binding to a part of the 100th base sequence from the 5 'end of the cell free fetal mRNA to be present in the blood, 'Obtaining a cDNA consisting of a sequence complementary to the RNA consisting of 20 to 100 base sequences consecutive from the end; And

(c) identifying amplification products obtained by performing a polymerase chain reaction with a primer set capable of amplifying the cDNA.

Hereinafter, each step of the present invention will be described in detail.

(a) obtaining blood collected from the mother

Step (a) is a step of obtaining blood collected from the mother, wherein the blood contains cell free fetal nucleic acid. The blood can be obtained without limitation the blood collected from the mother by the method of directly collecting blood from the mother, the method of purchasing blood collected in advance.

As used herein, the term "cell free fetal DNA or RNA (cff DNA or RNA), hereinafter referred to as" fetal nucleic acid "means free from cells known to be present in maternal blood (plasma and serum). Refers to a truncated nucleic acid, ie, a DNA or RNA fragment, and is known to consist of approximately 100 bases. Fetal rhesus D (RhD) genotyping by detecting fetal nucleic acid in maternal plasma or serum using techniques based on polymerase chain reaction (PCR) (Lo et al, N. Engl. J. Med. 339: 1734-1738, 1998), fetal gender tests (see Costa et al, N. Engl. J. Med. 346: 1502, 2002), and various fetal diseases can be diagnosed. Are known (Amicucci et al, Clin. Chem. 46: 301-302, 2000; Saito et al, Lancet 356: 1170, 2000); and Chiu et al, Lancet 360: 998-1000, 2002 ]).

The mother may be a pregnant woman having a gestation period suitable for the investigation of the gene expression pattern of the fetus through the detection result of the fetal nucleic acid using the method of the present invention. Suitable timing of conception may vary depending on the disease associated with the fetal nucleic acid to be detected.

Blood collection from the mother may be performed according to standard protocols generally followed by hospitals or clinics.

Detection of fetal nucleic acid found in maternal blood in the present invention can be performed using, for example, whole blood, serum or plasma. Methods for preparing serum or plasma from maternal blood are well known to those skilled in the art. For example, to prevent blood coagulation, the blood of a pregnant woman may be placed in a test tube containing EDTA or in a special commercial product, such as Vacutainer, and then centrifuged to obtain plasma from whole blood. Can be. On the other hand, serum can be obtained without centrifugation after blood coagulation.

In the present invention, in order to further improve the detection efficiency of fetal nucleic acid present in maternal blood, it may further comprise the step of extracting the nucleic acid from the obtained blood. Numerous methods for extracting nucleic acids from biological samples, including blood, are known, and nucleic acids can be obtained from blood samples taken from pregnant women using commercially available reagents or kits.

(b) cDNA Obtaining

Step (b) is a process of reverse transcription of the fetal mRNA into cDNA for detection and quantification of fetal mRNA present in maternal blood. Specifically, 20 to 100 consecutive from the 5 'end of the mRNA using a reverse transcription primer capable of complementarily binding to a portion of the 100th base sequence from the 5' end of the fetal mRNA to be present in the blood Obtaining cDNA consisting of a sequence complementary to RNA consisting of two base sequences.

As used herein, the term "primer" refers to a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free 3 'terminal hydroxyl group and serving as a starting point for template strand copying. .

As used herein, the term "reverse transcription primer" refers to a nucleic acid sequence having a short free 3 'terminal hydroxyl group, which is capable of forming a base pair with complementary template RNA and serving as a starting point for template strand copying. It means a nucleic acid sequence used for synthesis.

The primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.

In the present invention, synthesized cDNA for RNA having the shortest possible length (consisting the smallest number of sequences) from the 5 'end of the fetal mRNA, and after obtaining the amplification product through the polymerase chain plaque, Characterized in that detection, step (b) is the step of synthesizing cDNA for this.

In one embodiment of the present invention, after synthesizing each of the cDNA of different lengths by using a reverse transcription primer specific for the position of 300nt, 200nt, 100nt and 60nt from the 5 'end of the hCG mRNA of the fetal mRNA to be detected, Real-time PCR was performed on cDNA to compare the detection efficiency. As a result, when real-time PCR was performed targeting a portion closer to the 5 'end of mRNA, it was confirmed that fetal nucleic acid detection efficiency could be improved. In addition, when the sequence of the reverse transcription primer for cDNA synthesis is short, it was confirmed that the detection efficiency of fetal nucleic acid can be improved more (Fig. 2 and Table 3).

The cDNA may be composed of a sequence complementary to RNA consisting of 20 to 100, preferably 20 to 80, more preferably 20 to 60 nucleotide sequences consecutive from the 5 'end of the fetal mRNA to be detected, This can be obtained using reverse transcriptase primers capable of complementarily binding to a portion of the 100th sequence from the 5 'end of fetal mRNA.

Preferably, reverse transcription primers consisting of 5 or 6 base sequences may be used to obtain the cDNA.

(c) Amplification products  Step to verify

Step (c) is a process of detecting fetal mRNA present in blood collected from the mother by identifying the amplification product obtained by performing PCR with a primer set capable of amplifying the cDNA fragment obtained in step (b).

As used herein, the term "detection" means not only confirming the presence of fetal nucleic acid present in maternal blood, but also confirming the amount thereof.

According to the present invention, it is possible to confirm the presence or absence of fetal nucleic acid, such as Y-chromosome-derived nucleic acid, present in the mother's blood, and to determine the sex of the fetus. Genetic diseases such as syndrome (Trisomy 21), Edwards syndrome (Trisomy 18), and Patau syndrome (Trisomy 13) can be diagnosed.

Primer sets available in the present invention can be easily used by using sequences of fetal RNA or DNA disclosed in GENBANK or the like to complementarily bind to the 3 'terminal portion of cDNA to amplify the cDNA obtained in step (b). Can be designed. The primers can be chemically synthesized using phosphoramidite solid support methods or other well known methods.

According to the present invention, the cDNA obtained in step (b) can detect fetal mRNA present in the blood by identifying the amplification product obtained by PCR, but the method for identifying the amplification product is not limited thereto. However, electrophoresis, preferably real time PCR methods can be used.

When using the real-time PCR method to identify the amplification products in the present invention, the step (c) is amplified by performing a real-time PCR using a primer set capable of amplifying the cDNA and a probe capable of complementarily binding to the cDNA The presence of the product and its amount can be confirmed in real time.

As used herein, the term "probe" refers to an oligonucleotide that can bind to a minimum portion of other oligonucleotides of interest.

Preferably, probes usable in the present invention may include fluorescent, radioactive and luminescent systems and may be labeled with a "report molecule" for detection in other detection systems. The invention is not limited to any particular detection system or label.

The probes usable in the present invention consist of base sequences that can complementarily bind to the cDNA fragments, and can be easily designed using sequences of fetal RNA or DNA disclosed in GENBANK.

Preferably, the probe of the present invention may be a hydrolysis probe. The hydrolysis probe is known as a TaqMan analyte, typically labeled at the 5 'end with a reporter and at the 3' end with a fluorescent inhibitor.

Preferably, the probe of the present invention may be labeled with a fluorescent material and a fluorescent inhibitor at the 5 'end and the 3' end, respectively.

Fluorescent material that can be labeled at the 5 'end may be used without limitation fluorescent materials commonly used in the art, for example, 6-carboxyfluorescein, hexachloro-6-carboxy Hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein, FAM (5-carboxy fluorescein), HEX (2 ', 4', 5 ', 7'- tetrachloro-6-carboxy-4,7-dichlorofluorescein) and Cy5 (cyanine-5) and the like, but the 5 'terminal fluorescent substance of the present invention is not limited by the above examples.

Fluorescent inhibitors that can be labeled at the 3 'end can also be used without limitation, fluorescent materials commonly used in the art, such as 6-carboxytetramethyl-rhodamine, TAMRA (5 -Carboxytetramethylrhodamine), BHQ 1, 2, 3 (black hole quencher 1, 2, 3) and the minor groove binder (MGB), etc., but the fluorescent inhibitors that can be used in the present invention by the above examples are not limited.

Preferably, the present invention may further comprise the step of performing an extension reaction with an extension primer comprising a sequence complementary to the 3 'end of the cDNA fragment after step (b). The extension may be performed for the purpose of designing a probe when the length of the cDNA obtained in step (b) is short, preferably 20 to 50, more preferably 20 to 30 nucleotide sequences. Preferably, the extension primer may include a hairpin structure (stem and loop) at the 5 'end.

In one embodiment of the present invention, cDNA consisting of a sequence complementary to the 20 consecutive sequences from the 5 'end of the hCG mRNA, synthesized, and attached to the end of the cDNA with a stem and loop primer, the real-time PCR The detection efficiency by was confirmed. As a result, it was possible to detect hCG more effectively than real-time PCR of cDNA consisting of sequences complementary to 100 consecutive sequences from the 5 'end of hCG mRNA, and confirmed that there was no variation between samples (Table 5). And FIG. 5).

As one preferred embodiment of the present invention, steps (a), (b) and (c) of the present invention are exemplarily described through a process of detecting hCG mRNA, which is one of fetal mRNAs present in maternal blood. Same as First, RNA is obtained by obtaining blood collected from a mother. Subsequently, cDNA complementary to 20 sequential sequences from the 5 'end of hCG mRNA is synthesized using a reverse transcript primer consisting of 6 mer nucleotide sequences represented by SEQ ID NO: 20. Subsequently, the hairpin structure was attached to the 3 'end of the cDNA using a stem and loop primer represented by SEQ ID NO: 21, and then the forward and reverse primers represented by SEQ ID NO: 22 or 23, and the 5' end were labeled with FAM. Perform real-time PCR using the probe indicated by number 24. By measuring the fluorescence signal emitted during the PCR process and quantifying the hCG mRNA present in the mother's blood, the expression pattern of the fetal gene can be investigated.

In another embodiment, the present invention provides a method for producing blood, comprising the steps of: (a) obtaining blood collected from a mother; And (b) polymerase chain reaction with a primer set capable of complementarily binding to a portion of the 100th base sequence from the 5 'end of the cell free fetal DNA to be present in the blood. It provides a method for detecting the nucleic acid of the fetus in the mother's blood, comprising the step of identifying the amplification product consisting of 20 to 100 consecutive sequences from the 5 'end of the DNA obtained by.

Regarding the description of each step of the present invention, the same parts as those described above or parts readily applicable to those skilled in the art are omitted to avoid repetition.

Step (a) is a step of obtaining blood collected from the mother, as described above.

The step (b) is performed by polymerase chain reaction with a primer set capable of complementarily binding to a part of the 100th nucleotide sequence from the 5 'end of the fetal DNA to 20 to 100 consecutive from the 5' end of the DNA. Obtaining an amplification product consisting of three nucleotide sequences, the amplification product is confirmed by the process of detecting fetal DNA present in the blood collected from the mother. The fetal DNA can be detected early to detect fetal sex or genetic diseases.

The amplification product may consist of 20 to 100 contiguous, preferably 20 to 80, more preferably 20 to 60 base sequences from the 5 'end of the fetal DNA to be detected, which is 5' of fetal DNA. It can be obtained by using a set of primers capable of complementarily binding to a portion of the 100th base sequence from the end.

Identification of the amplification product can be confirmed using electrophoresis, preferably real-time PCR method, the matters related to real-time PCR is as described above.

Prenatal screening due to low fertility and aging is preferred. So far, amniotic fluid testing has been dangerous and inaccurate, with half of mothers refusing to test. The present invention can detect fetal nucleic acid present in the blood safely and with high accuracy, and can be usefully used to identify and treat genetic analysis and disease of the fetus in a manner that is not dangerous to both mother and fetus.

1 is a schematic diagram showing the length and location of RNA used to find a method for effectively detecting cell free fetal RNA.
(1) is a schematic diagram showing the cDNA synthesis position of 300nt, 200nt, 100nt, 60nt from the 5 'end of hCG mRNA.
(2) is a schematic diagram to confirm the detection difference of the cell free fetal RNA according to the position of the 5 'end and the middle and 3' end during cntNA synthesis of 100nt length
(3) is a schematic diagram showing the location of 5SLAM (5 'stem and loop amplification method).
Figure 2 is a graph showing the detection efficiency of hCG mRNA according to the length from the mRNA 5 'end.
Figure 3 is a graph showing the comparison results of the detection efficiency of hCG mRNA for each position at the 5 'end, the middle and 3' end. The closer it is to the 5 'end, the lower the Cq value, indicating that it can be detected more effectively.
4 is a schematic diagram of a 5SLAM process.
5 shows a comparison result of the detection efficiency of hCG mRNA of 5AM (5 'amplification method) and 5SLAM of 100nt.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Example  One : mRNA Comparison of the Detection Efficiency According to the 5 'End Positions of-5AM (5' amplification method )

RNA was isolated from plasma and each cDNA of different length using specific reverse transcription primers (Table 1) at 300nt, 200nt, 100nt and 60nt from 5 'end of hCG mRNA Was synthesized. When synthesizing 300nt, 200nt, 100nt, 60nt cDNA, 15 mer reverse primer was used as RT primer and 60nt further reacted with shorter 5 mer RT primer. It was. Thereafter, 50 ng of the cDNA was subjected to real-time PCR with a hydrolysis probe (Table 1) equipped with FAM to confirm the detection efficiency according to the length of the 5 'end, and the results are shown in FIG. 2. PCR proceeded with a total of 20 μl reaction, using 10 pmol of forward and reverse primers and 5 pmol of hydrolysis probe, 5 nmol dNTP mix, 75 mM Tris HCl (pH 9.0), 2 mM MgCl ₂ , 75 mM KCl PCR was performed with Taqpolymerase (Biotools, Madrid, Spain) at 30 mM (NH ₄ ) ₂ SO ₄ , 1U. At this time, the reaction was amplified by 40 cycles of 95 ℃, 5 min, 95 ℃, 15 sec, 52 ℃, 1 min.

As shown in Figure 2, the detection efficiency was the best when the length from the 5 'end of the detection target mRNA is 60nt. In addition, the RT primer was superior to the detection efficiency when using a primer of 5 to 6 mer length than when using the reverse primer of 15 mer length (Fig. 2).

Through the above results, fetal nucleic acid can be detected with higher efficiency when targeting the portion close to the 5 'end of the detection target mRNA, and it can be confirmed that the shorter the length of the primer can improve the detection efficiency. there was.

Example  2 : mRNA  Comparison of Detection Efficiency by Location

The closer to the 5 'end of the target nucleic acid, the smaller the size of the amplification, the better the detection efficiency after confirming through Example 1, short cut at the position of the mid (mid) and 3' end (3 'end) of the total mRNA It was confirmed whether the detected mRNA fragment (100nt) can be effectively detected compared to the 5 'end. CDNA was synthesized using 6 mer RT primers capable of specific reverse transcription of mRNA at each position, and real-time PCR was performed using forward and reverse primers and probes specifically prepared for each cDNA (Table 2). ), And the results are shown in Figure 3 and Table 3. PCR proceeded with a total of 20 μl reaction, using 10 pmol of forward and reverse primers and 5 pmol of hydrolysis probe, 5 nmol dNTP mix, 75 mM Tris HCl (pH 9.0), 2 mM MgCl ₂ , 75 mM KCl PCR was performed with Taqpolymerase (Biotools, Madrid, Spain) at 30 mM (NH ₄ ) ₂ SO ₄ , 1U. At this time, the reaction was amplified by 40 cycles of 95 ℃, 5 min, 95 ℃, 15 sec, 60 ℃, 1 min.

As shown in FIG. 3 and Table 3, when real-time PCR of the same length cDNA, it was confirmed that the closer to the 5 'end, the lower the Cq value can be detected more efficiently. That is, cff RNA remaining in the plasma was degraded (degradation) it was confirmed that a lot of short fragments of the 5 'end remains (Fig. 3 and Table 3).

The above results indicate that the cff nucleic acid can be detected more efficiently when targeting the 5 'end than when targeting the middle or 3' end.

Example  3: 5SLAM (5 ' stem and loop amplification method )

Through the results of Examples 1 and 2, it was confirmed that the smallest cleaved cff nucleic acid can be detected as close as possible to the 5 'end of mRNA and maximized when amplified to a small size. Accordingly, a method (5SLAM, 5 'stem and loop amplification method) was designed to detect cff RNA with only 20nt of the smallest size that can be as close as possible to the 5' end. 4 is a schematic diagram of the 5SLAM method, and Table 4 shows the sequences of primers and probes used for 5SLAM.

To summarize the 5SLAM method, cDNA was synthesized using a 6 mer long specific RT primer at 20 nt of hCG mRNA, and has a sequence capable of complementarily binding to 6 mer at the 3 'end of the product. DNA with stem and loop tails was synthesized by extension with stem and loop primers. Thereafter, using a hydrolysis probe having a stem and loop position sequence and a hCG sequence as a target gene, the hydrolysis probe is broken by forward and reverse primers to generate g-fluorescence, and the result is illustrated in FIG. 5. And in Table 5. PCR proceeded with a total of 20 μl reaction, using 10 pmol of forward and reverse primers and 5 pmol of hydrolysis probe, 5 nmol dNTP mix, 75 mM Tris HCl (pH 9.0), 2 mM MgCl ₂ , 75 mM KCl PCR was performed with Taqpolymerase (Biotools, Madrid, Spain) at 30 mM (NH ₄ ) ₂ SO ₄ , 1U. At this time, the reaction was amplified by 40 cycles of 95 ℃, 5 min, 95 ℃, 15 sec, 57 ℃, 1 min.

As shown in FIG. 5 and Table 5, the 5SLAM method synthesized 100 nt cDNA at the 5 'end, was amplified faster than the real time PCR method (5AM), and confirmed that the variation was less. (FIG. 5). These results indicate that the 5SLAM method can effectively detect short cleaved cff RNA in maternal blood, in which case the variation between samples is less effective.

Attach an electronic file to a sequence list

Claims (11)

(A) obtaining a cDNA complementary to the RNA fragment consisting of 20 to 100 sequential sequences from the 5 'end of the cell free fetal mRNA in the blood taken from the mother, the cDNA is Obtained by reverse transcription using a primer complementary to the 5 'end of the cell free fetal mRNA and a primer complementary to a portion of the 20th to 100th base sequence from the 5' end of the cell free fetal mRNA;
(b) performing an extension reaction with an extension primer having a stem and loop at the 5 'end, comprising a sequence complementary to the 3' end of the cDNA fragment; And
(c) identifying the amplification product obtained by performing a polymerase chain reaction with a primer set capable of amplifying the cDNA.
The method of claim 1,
The primer used for reverse transcription of step (a) is composed of 5 or 6 base sequences.
The method of claim 1,
Step (a) is a step of obtaining a cDNA complementary to the RNA fragment consisting of 20 to 60 consecutive sequences from the 5 'end of the cell free fetal mRNA, the cDNA is 5' end of the cell free fetal mRNA Obtained by reverse transcription using a primer complementary to and a primer complementary to a portion of the 20th to 60th nucleotide sequence from the 5 'end of the cell free fetal mRNA.
The method of claim 1,
Identification of the amplification product in step (c) is confirmed by performing a real-time polymerase chain reaction using a primer set capable of amplifying the cDNA and a probe capable of complementarily binding to the cDNA.
delete delete (a) a complementary primer to the 5 'end of cell free fetal DNA in the blood taken from the mother and a portion of the 20th to 100th base sequence from the 5' end of the cell free fetal DNA; Identifying an amplification product consisting of 20 to 100 base sequences consecutively obtained from the 5 'end of the DNA obtained by performing a polymerase chain reaction with a primer; And
(b) further performing an extension reaction with an extension primer having a hairpin structure (stem and loop) at the 5 'end, comprising a sequence complementary to the 3' end of the amplification product obtained in (a) above; To detect the nucleic acid of the fetus in the mother's blood.
8. The method of claim 7,
Step (a) is performed by using a primer complementary to the 5 'end of the cell free fetal DNA and a primer complementary to a portion of the 20th to 60th nucleotide sequence from the 5' end of the cell free fetal DNA. 'To obtain an amplification product consisting of 20 to 60 sequential sequences consecutive from the end.
8. The method of claim 7,
Identification of the amplification product in the step (a), it is confirmed by performing a real-time polymerase chain reaction using a probe capable of complementary binding to the amplification product.

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