CN111020022B

CN111020022B - Method and kit for detecting chromosomal rearrangements

Info

Publication number: CN111020022B
Application number: CN201910710269.1A
Authority: CN
Inventors: 丁春明; 金胜男; 尚庆娟
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2019-08-01
Filing date: 2019-08-01
Publication date: 2020-12-29
Anticipated expiration: 2039-08-01
Also published as: WO2021017543A1; CN111020022A

Abstract

The present invention relates to a method and a kit for detecting chromosomal rearrangements. Specifically, the present invention relates to a method for detecting a chromosomal rearrangement, comprising obtaining sample DNA from a sample from a subject; long-range PCR (LD-PCR) and quantitative PCR (qpcr) were performed on the sample DNA; and comparing the test results with the results of a normal control sample; and deducing from the result of the comparison whether a chromosomal rearrangement is present in the sample DNA. The LD-PCR and qPCR may be performed sequentially or in the same reaction vessel. The invention also relates to kits and primers and probes for performing said methods.

Description

Method and kit for detecting chromosomal rearrangements

Technical Field

The invention relates to the field of biotechnology, in particular to a method and a kit for detecting chromosome rearrangement, such as chromosome segment deletion, duplication, inversion, translocation and the like. The invention also specifically relates to a method and a kit for detecting the inversion of the 22 # intron of the F8 gene.

Background

A chromosomal rearrangement is a chromosomal abnormality that involves a change in the native chromosomal structure. The alterations may involve several different types, e.g., deletions, duplications, inversions, translocations, etc. Typically, these changes are due to the disruption of two different positions in the DNA double helix, followed by rejoining of the disrupted ends to produce a new chromosomal arrangement of genes that differs in gene order from the chromosomes prior to disruption. Chromosomal rearrangements can cause various diseases.

Hemophilia a is an X-linked recessive inherited bleeding disorder. The incidence in the male population is 1/5000. Due to the fact that the F8 gene mutation causes insufficient FVIII coagulation factor content or quality defect, endogenous coagulation pathway disorders in which FVIII coagulation factors participate are caused, and bleeding is caused. Wherein, the inversion of the 22 intron of the F8 gene is the main pathogenic factor of patients suffering from hemophilia A. Therefore, the method is crucial to the detection of the inversion of the 22 th intron.

At present, the methods for detecting the inversion of the 22 # intron mainly comprise southern blot hybridization, LD-PCR, I-PCR, IS-PCR and the like.

Southern blot hybridization was performed using gold standards for Inv22 detection. Firstly, digesting the genome DNA of a patient to be detected by Bcl restriction endonuclease, and then, carrying out agarose gel electrophoresis to separate DNA fragments; carrying out alkali treatment on the gel to denature DNA, and then, imprinting the denatured DNA onto a nitrocellulose filter membrane or a nylon membrane from the gel; the analysis is carried out by hybridizing a DNA probe labeled with radioactivity or non-radioactivity to DNA on a solid support, and displaying a hybridization band by a corresponding method according to the labeling characteristics of the probe. The radioactive or non-radioactive labeled DNA probes can bind to int22h-1, int22h-2 and int22 h-3. Wild-type patients will show 3 bands of 14kb, 16kb and 21.6 kb; patients with intron 22 inversion type 1 showed 3 bands of 14kb, 17.5kb and 20kb, respectively; patients with intron 22 in inverted 2 form presented 3 bands of 15.5kb, 16kb and 20kb, respectively. Southern blot hybridization not only distinguished the wild type from the intron 22 inversion, but also recognized the Inv22 type. But the operation is complicated, the result needs 8 to 10 days, and the time is consumed; large amount of DNA, potential radioactive pollution and the like.

In 1998, Liu et al designed long-range PCR (LD-PCR) to detect inversion of intron 22 of F8 gene. 4 primers P, Q, A, B were designed to distinguish wild type, inverted and carrier by detecting the inversion directly from genomic DNA in the same reaction tube. Two primers P and Q are located at 1212bp and 1224bp of int22h-1 flanking sequence in FVIII gene respectively. Two primers A and B are located at int22h2/3 flanking sequences 162bp and 118bp, respectively. When male hemophilia patients are detected, if a PQ fragment of 12kb and an AB fragment of 10kb are generated, the male hemophilia patients are wild; if the PB + AQ fragment of 11kb and the AB fragment of 10kb are inverted; when the female was examined, 3 bands of PQ fragment of 12kb, PB + AQ fragment of 11kb and AB fragment of 10kb were generated, and the female was the carrier. In all cases, the AB segment serves as an internal control, since at least one homologous sequence of int22h-2 or int22h-3 remains unchanged. The three amplified fragments were distinguished by electrophoresis on a 0.6% agarose gel. Because the method amplifies the fragments with longer length (10-12kb) and the GC island with 3.5kb in the amplified fragments, the requirements on the amount of DNA and the integrity of a DNA template are higher. LD-PCR has higher requirements on the concentration and quality of leukocyte DNA; meanwhile, when the LD-PCR amplification product is identified through the result of agarose gel electrophoresis, the sensitivity is influenced by the resolution of the agarose gel, and the detection range is limited. LD-PCR is carried out for 30 cycles, which takes 6-7 hours; meanwhile, agarose gel electrophoresis is needed for result judgment, so that the operation is complex, and the agarose gel electrophoresis is not favorable for clinical application.

The AQ-PLP (AccuCopy qualification combined with pre-amplification of long-distance PCR) technology firstly utilizes 2 individual systems to respectively carry out 12 cycles LD-PCR, a patient system 1 without inversion obtains a fragment containing M1, and a patient system 2 obtains a fragment containing M3; inv 22-type I inverted patient System 1 gave a fragment containing M3 and System 2 gave a fragment containing M1; inv 22-type II inverted patient System 1 gave a fragment containing M2 and System 2 gave a fragment containing M1. And then carrying out multiple fluorescent competitive PCR amplification, and identifying the obtained amplification product through fluorescent capillary electrophoresis to finally obtain the inversion type of the hemophilia patient. The method has complex experimental design and complex result judgment. In addition, the experimental steps are more, the operation is complicated, and the pollution of a laboratory can be caused when the multiple fluorescence competitive PCR amplification is carried out on the 12-cycle LD-PCR product.

In 2005, Rossetti et al designed inverse PCR to detect inversion of intron 22 of F8 gene, which included three steps: firstly, cutting genome DNA by BclI restriction endonuclease; secondly, connecting the BclI digested fragments into a ring by using ligase; in the third step, three primers IU, ID and ED are used to perform a standard multiplex trans PCR reaction. For the 22 # intron inverted wild type, a 487bp fragment was generated, whereas a 559bp fragment was amplified in hemophilia patients of Inv22, and two fragments were amplified in female Inv22 carriers: 487bp and 559 bp.

In 2008, Rossetti et al improved on the basis of inverse PCR, forming a new detection method: inverse shift PCR (Inverse shift-PCR, IS-PCR). IS-PCR IS a variant of inverse PCR, a method by which restriction enzyme fragments can be detected, and relies on specific primers directed against terminal sequences in the restriction enzyme sites. Thus, changes in the size of the IS-PCR product fragment ultimately reflect specific changes in the DNA nucleotide composition at the end of the restriction fragment. IS-PCR consists of two parts, one for diagnostic assays and the other for complementary assays. This genotyping strategy may be applicable to all known Inv22 types in the human genome. The diagnosis experiment consists of four primers consisting of 1U, 2U and 3U, ID, and the supplementary experiment consists of 1U, 2U and 3U, ED. The Inv22 can be distinguished according to different amplified fragments and different fragment lengths, and the diagnosis experiment can detect dup22 as a wild-type patient and del22 as an Inv22 inverted patient, but because dup22 is a non-pathogenic mutation and del22 is a pathogenic mutation, the Inv22 diagnosis test can distinguish an HA pathogenic mutation from a non-HA pathogenic mutation; further supplementary tests were carried out to complement the differences between Inv22 and Del22 and Dup22 and Normal, and to detect almost all known rearrangements. However, the method has the defects of complex operation and high technical requirement; and the restriction fragments are too long to form a ring.

Thus, there remains a need in the art for a simple, rapid, sensitive, and accurate method for detecting F8 Inv 22.

Disclosure of Invention

The present invention meets the above needs by the following technical solutions:

in one aspect, the invention relates to a method for detecting a chromosomal rearrangement comprising the steps of:

a. obtaining sample DNA from a sample from a subject;

b. performing long-range PCR (LD-PCR) on the sample DNA;

c. carrying out quantitative PCR (qPCR) on the product obtained by the LD-PCR; and

d. comparing the results of step c with the results of a normal control sample; and deducing from the result of the comparison whether a chromosomal rearrangement is present in the sample DNA.

In a particular embodiment, the present invention relates to a method for detecting a chromosomal rearrangement comprising the steps of:

a. obtaining sample DNA from a sample from a subject;

b. performing long-range PCR (LD-PCR) on the sample DNA using primers designed for the normal chromosomal region and primers designed for the rearranged chromosomal region; if the chromosome is normal, amplifying to obtain a normal amplified fragment, and if the chromosome has rearrangement, obtaining an amplified fragment of a rearrangement region;

c. performing quantitative pcr (qpcr) on the normal amplified fragment of step b and the amplified fragment of the rearranged region of step b, respectively; and

d. and c, if the qPCR signal of the amplified segment aiming at the rearrangement region is enhanced relative to the qPCR signal of the normal control in the step c, judging that the chromosome rearrangement occurs.

The chromosomal rearrangement may be, for example, an inversion of intron 22 of the F8 gene.

In one embodiment, step b uses SEQ ID NO: 1-3. In one embodiment, step c uses SEQ ID NO:4-7 and the primers of SEQ ID NO:8 and 9.

In another aspect, the invention relates to a method for detecting a chromosomal rearrangement comprising the steps of:

a. obtaining sample DNA from a sample from a subject;

b. performing LD-PCR and qPCR on sample DNA in a single reaction vessel; and

c. comparing the results of step b with the results of a normal control sample; and deducing from the result of the comparison whether a chromosomal rearrangement is present in the sample DNA.

a. obtaining sample DNA from a sample from a subject;

b. performing long-range PCR (LD-PCR) on the sample DNA in a single reaction vessel using primers designed for the normal chromosomal region and primers designed for the rearranged chromosomal region; if the chromosome is normal, amplifying to obtain a normal amplified fragment, and if the chromosome has rearrangement, obtaining an amplified fragment of a rearrangement region; performing quantitative pcr (qpcr) on the normal amplified fragment and the amplified fragment of the rearranged region, respectively; and

c. and (c) judging that the chromosome rearrangement occurs if the qPCR signal of the amplified segment aiming at the rearrangement region in the step (b) is enhanced relative to the qPCR signal of the normal control.

In one embodiment, wherein step b uses SEQ ID NO:1-3 and SEQ ID NO:10-13 and the primers of SEQ ID NO:8 and 9.

The sample used in the method of the present invention may be a body fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, stool and tissue sample, and the like.

In one embodiment, LD-PCR is performed for 5-20 rounds, for example, may be 12 rounds.

The invention also relates to a kit for detecting chromosomal rearrangements, comprising primers for performing LD-PCR on sample DNA from a subject and primers and probes for performing qPCR on the products of said LD-PCR.

In one embodiment, the primers and probes for performing LD-PCR and the primers and probes for performing qPCR are placed in separate containers. In one embodiment, the primers used to perform LD-PCR are SEQ ID NO:1-3, and the primers and probes used to perform qPCR are SEQ ID NOs: 4-7 and SEQ ID NO: 8-9.

In another embodiment, the primers and probes for performing LD-PCR and the primers and probes for performing qPCR are placed in the same vessel. In one embodiment, the primers used to perform LD-PCR are SEQ ID NO:1-3, and the primers and probes used to perform qPCR are SEQ ID NOs: 10-13 and SEQ ID NO: 8-9.

The kit may further comprise instructions for performing the methods of the invention.

In another aspect, the invention also relates to novel primers comprising a sequence selected from the group consisting of SEQ ID NO:4-7 and SEQ ID NO:10-13 nucleotide sequence.

In another aspect, the invention relates to a novel probe comprising SEQ ID NO:8 or 9.

The invention realizes the combined use of LD-PCR and dual fluorescent quantitative PCR (qPCR) technology for the first time. Firstly, three primers P, Q and 2F3R are used for carrying out 12-cycle LD-PCR, and the P & Q fragments are obtained by amplifying the P & Q of patients without inversion; the patients with the inversions are amplified between the P &2F3R to obtain a P &2F3R fragment; amplification occurred between inverted carriers P & Q, resulting in P & Q fragments, and between P &2F3R, resulting in P &2F3R fragments.

Then, double real-time fluorescent quantitative PCR is carried out, and a pair of specific primers and probes are designed on the P & Q fragments: m1 primer and M1 probe; designing a pair of specific primers and probes on the P &2F3R fragment: m2 primer and M2 probe. Thus, when qPCR is performed, the amount of M1 may indirectly reflect the amount of P & Q fragments; the amount of M2 may indirectly reflect the amount of P &2F3R fragment. Because the P & Q fragment of the patient without inversion is amplified and the P &2F3R fragment is not amplified, the template amount of M1 is larger than that of M2 when qPCR is carried out; on the other hand, the P &2F3R fragment of the patient in the inverted position was amplified, and the P & Q fragment was not amplified, so that the amount of the M2 template was greater than that of the M1 template during qPCR.

By utilizing 3 primers to carry out LD-PCR of 12 cycles and combining a qPCR method to detect the contents of two sites M1 and M2, 30 cycles are shortened to 12 cycles on the basis of the common LD-PCR, thereby greatly saving the LD-PCR process which consumes long time; meanwhile, the complicated agarose gel process is removed, the advantages of high flux, high sensitivity and the like of qPCR are combined, the inversion of the No. 22 intron of the F8 gene is detected, and the detection method is more sensitive.

The invention firstly establishes the methodology of the LD-PCR combined qPCR two-step method. However, in the two-step experiment, when the 12-cycle LD-PCR product is subjected to qPCR, the product needs to be exposed in the air, and laboratory pollution can be caused, so that the one-step optimization is carried out on the basis of the two-step method.

Inv22 in male hemophilia patients and Inv22 carriers in female samples can be accurately, effectively and quickly screened by a one-step method of closed-tube nested quantitative PCR (CN-qPCR) combining LD-PCR and qPCR. In the one-step experiment, the detection cost of each patient leukocyte DNA sample is less than 10 yuan; the time of the whole experiment is 4.5 h; the amount of leukocyte DNA used per sample is small and the appropriate concentration range for the amount of DNA template is wide (e.g., 10-80ng, particularly 10-40 ng).

The advantages of the one-step method, such as simplicity, rapidness, low cost, high sensitivity, small DNA dosage and the like, are more favorable for clinically and rapidly screening the inversion position of the intron 22 of the F8 gene. All reaction systems are completed by one-time sample adding, and then the tube opening is not needed, so that the operation is simple. First, 12 cycles are carried out through LD-PCR, which greatly reduces the time required in the common LD-PCR experiment; meanwhile, the amplified product is directly subjected to qPCR to detect F8 Inv22, so that the detection sensitivity is improved, and the complicated steps of agarose gel electrophoresis are removed, so that the experiment is more favorable for clinical application. The invention can accurately, effectively and quickly screen Inv22 in male hemophilia patients and Inv22 carriers in female samples.

The skilled person will appreciate that the principles of the above method are not only applicable to the detection of Inv22 of the F8 gene in hemophiliacs. Other chromosomal rearrangements, such as chromosomal segment deletions, duplications, inversions, translocations, fusions, etc., can also be detected using the LD-PCR combined qPCR method described above according to the same principles.

In the rearrangement at the chromosome level, the related breakpoint is generally in a gene region, and the region is generally large (the minimum possible is several hundred bp, a little bit is several kb, or even larger), so long-distance PCR (LD-PCR) is often required for amplifying the rearrangement region, and the long-distance PCR is time-consuming, has low amplification efficiency, and the product generally needs to be identified by electrophoresis, is troublesome and easily causes aerosol pollution.

In the invention, the LD-PCR only needs to be carried out for a limited number of cycles without being carried out completely, thereby saving time; the LD-PCR product is directly quantified through qPCR, the sensitivity is high, and the quantification can be realized; in addition, the single tube closed reaction greatly reduces the aerosol pollution risk.

Drawings

FIG. 1 shows the experimental principle and the experimental results of a two-step method combining LD-PCR and qPCR. A: test results for non-Inv 22 male hemophiliacs; b: inv22 results of testing male hemophiliacs.

FIG. 2 shows the experimental principle and the experimental results of the LD-PCR combined qPCR one-step method. A: test results for non-Inv 22 male hemophiliacs; b: inv22 results from a test on male hemophiliacs; c: non-Inv 22 test results; d: inv22 test results from female carriers.

FIG. 3 shows LD-PCR in combination with qPCR one-step method for the detection of male hemophilia patients and female carriers. A: 80 patients with hemophilia A with different degrees of severity; b: results of testing of 10 Inv22 female carriers and 10 non-Inv 22 women.

FIG. 4 shows a combination of LD-PCR and qPCR one-step method for the detection of male hemophilia patients with different amounts of substrate DNA.

FIG. 5 shows a schematic diagram of the detection of chromosomal microdeletions (e.g., 1p36 deletion).

Fig. 6 shows a schematic diagram of fusion detection of ALK gene and EML4 in lung cancer. As shown, a particular fusion site may be fused at a point between intron 5 and intron 6 of EML4, as well as at a point in intron 19 of ALK. Multiple forward primers, each about 4kb apart, can be designed in the region of intron 5 and intron 6 of EML4, and then a reverse primer can be designed in the region of intron 19 of ALK for large-fragment PCR amplification. Two qPCR were designed with fluorescence 1 within the fusion region (the region where the reverse and forward primers can amplify) and fluorescence 2 outside the fusion region. After large-fragment PCR amplification, double fluorescence qPCR is carried out, and the signal of fluorescence 1 is greatly stronger than that of fluorescence 2, so that fusion occurs.

Detailed Description

Collection of samples used in the examples

Blood samples from hemophiliacs were collected at the second subsidiary hospital of the university of medical, wenzhou, with informed consent. Blood samples from female carriers and healthy women were collected at rekins hospital with informed consent. The sample collection personnel are professional blood collection nurses in hospitals, and the collection process is strictly carried out according to the principle of aseptic operation.

And placing the collected blood sample into a 5mL blood collection tube anticoagulated by sodium citrate or EDTA, and temporarily storing the blood sample in a refrigerator at 4 ℃ after the blood collection is finished. Storing in a refrigerator at-80 deg.C within 3 days after blood sampling.

Extraction of leukocyte DNA

Extraction of leukocyte DNA was performed using QIAamp DNA Blood Mini Kit (Qiagen).

Example 1

LD-PCR combined qPCR two-step method

Experimental procedures

The experiment was divided into two steps: the first step was 12 cycles of LD-PCR, the second step was 12 cycles of LD-PCR product and the original DNA without 12 cycles of LD-PCR was subjected to M1 and M2 site qPCR.

Samples of hemophilia patients were selected and systematically formulated as per the following table.

TABLE 1-1

Tables 1 to 2

Primer and probe sequences

Tables 1 to 3

12-cycle LD-PCR reaction solution composition

After the reaction system of the sample is configured, the sample is put into a Bio-Rad S1000 Thermal Cycler PCR instrument for PCR reaction. The procedure is as follows: at 98 ℃ for 1 min; 10s at 98 ℃ and 10min at 68 ℃ (+20 s/cycle), and 12 cycles are carried out; 10min at 72 ℃; keeping at 4 ℃. See tables 1-4 below.

Tables 1 to 4

After the completion of 12 cycles of LD-PCR reaction, 2. mu.L of 12 cycles of LD-PCR product and 2. mu.L of unamplified gDNA diluted 10 times were prepared according to the system shown in tables 1-5, respectively.

Tables 1 to 5

Composition of qPCR reaction solution

After the reaction system of the sample is prepared, the sample is put into a Bio-Rad C1000 Touch Thermal Cycler CFX 96Q-PCR instrument. The Bio-Rad CFX Manager3.1 software was turned on, and after selecting the Probe program in PrimePCR, the program was set and the plate was set, respectively. The procedure is as follows: at 95 ℃ for 10 min; 45 cycles of 95 ℃ for 15s, 59 ℃ for 30s and 72 ℃ for 30 s. See table below. When the plate is set, two fluorescence detection channels of FAM and HEX are selected, and the position of the selected sample is detected. After all the settings were completed, "Start Run" was clicked on to Start the qPCR reaction.

Tables 1-6 qPCR reaction procedure

Analysis of detection results

After the qPCR experiment was completed, the threshold for "FAM" was 97.69 and for "HEX" 98.53. Ct values of M1 and M2 obtained from the samples were analyzed.

Diagnosis of clinical sample hemophilia A male patient by LD-PCR combined qPCR two-step method

14 male patients with severe hemophilia a were tested in a two-step procedure, all samples were verified by gold standard LD-PCR and the test results are summarized in table 2. The Ct (M1-M2) threshold for patients with and without 22 intron inversions was 0. If Ct (M1-M2) < 0, Inv22 does not occur; if Ct (M1-M2) > 0, the male patient is declared to have Inv 22.

Therefore, in 14 male severe hemophilia A patients, inversion of the 22 th intron of the F8 gene was detected by a two-step method combining LD-PCR and Q-PCR, wherein 5 th intron inversion of the 22 th intron occurs and 9 th intron inversion of the 22 th intron does not occur. Consistent with the gold standard results.

TABLE 2

Note: LD-PCR-: the detection result of the original sample DNA of the LD-PCR is not carried out for 12 cycles; LD-PCR +: the results of 12 cycles of LD-PCR detection were performed.

See also figure 1 for the procedure and results of this experiment.

Example 2

LD-PCR combined qPCR one-step method

Experimental procedures

Samples to be tested were systematically prepared according to tables 3-1, 3-2 and 3-3. After the reaction system of the sample is prepared, the sample is put into a Bio-Rad C1000 Touch Thermal Cycler CFX 96Q-PCR instrument. The Bio-Rad CFX manager3.1 software was turned on, and after selecting the Probe program in PrimePCR, the program was set and the plate was set, respectively. The programming was performed according to tables 3-4. When the plate is set, two fluorescence detection channels of FAM and HEX are selected, and the position of the selected sample is detected. After all the settings were completed, "Start Run" was clicked on to Start the qPCR reaction.

TABLE 3-1 Main Agents

TABLE 3-2 primer and Probe sequences

TABLE 3-3 reaction solution Components

TABLE 3-4 reaction procedure

Analysis of detection results

Diagnosis of clinical sample hemophilia A male patient by LD-PCR combined qPCR one-step method

80 male hemophilia A patients were tested by one-step method, all samples were verified by gold standard LD-PCR, and the test results are summarized in Table 4-1. The Ct (M1-M2) threshold for patients with inversion of intron 22 and patients without inversion of intron 22 was-2.0. That is, if Ct (M1-M2) > -2.0, inversion of the No. 22 intron occurs; if Ct (M1-M2) < -2.0, it indicates that the male patient has not inverted intron 22. Whether the sample is inverted or not is verified by a gold standard LD-PCR method.

The detection of 80 samples in the method obtains definite results, and only 72 samples are detected by the conventional LD-PCR method.

We analyzed 72 patients detected by both methods in comparison, with 100% consistency. Finally, according to the results of LD-PCR combined Q-PCR, 18 of the 80 hemophiliacs had 22 intron inversions, and the remaining 62 patients had no 22 intron inversions; of these, 16 of 46 severe hemophiliacs had an inverted intron 22 (34.8%), 1 of 16 moderate hemophiliacs had an inverted intron 22 (6.3%), 1 of 11 mild hemophiliacs had Inv22 (9.1%), and 7 of unknown severity hemophiliacs had no Inv 22.

TABLE 4-1

Note: non-Inv 22 indicates that no inversion of the 22 intron of the F8 gene occurs; inv22 is the occurrence of inversion of intron 22 of F8 gene. NA is a hemophiliac of unknown severity.

The results are better than the gold standard. Conventional LD-PCR has high requirements for DNA templates, and sometimes results cannot be obtained when the quality of template DNA is poor or the amount of template DNA is insufficient. The method has high sensitivity, has low requirement on the DNA template, and greatly reduces the possibility of sample detection failure.

Diagnosis of clinical sample hemophilia A female carrier by LD-PCR combined qPCR one-step method

10 carriers of hemophilia a 22 female with inverted intron and 10 female healthy controls were tested by one-step method and the results are summarized in tables 4-2. The Ct (M1-M2) threshold for female carriers with inverted 22 introns versus female healthy controls was-3.0. That is, if Ct (M1-M2) > -3.0, it is the female carrier with inverted intron No. 22; if Ct (M1-M2) < -3.0, it indicates that the female sample does not carry the inversion of intron 22.

TABLE 4-2

See also fig. 2 and fig. 3 for the procedure and results of this experiment.

Example 3

One-step method for detecting inversion of intron 22 of F8 gene without influence of substrate DNA quantity

Experimental procedures

Analysis of detection results

Method for detecting hemophilia A samples with different concentrations by using LD-PCR combined qPCR one-step method

A total of 12 samples were obtained by detecting inversion of intron 22 on hemophilia A6 and inversion of intron 22 on hemophilia A6 by one-step method. Wherein the gDNA usage of each sample was 40ng, 20ng, 10ng, respectively. The results of the tests are summarized in Table 5. Under the condition of different substrate DNA amounts, the following conditions are also met: the Ct (M1-M2) threshold for patients with inversion of intron 22 and patients without inversion of intron 22 was-2.0. That is, if Ct (M1-M2) > -2.0, inversion of the No. 22 intron occurs; if Ct (M1-M2) < -2.0, it indicates that the male patient has not inverted intron 22.

TABLE 5

See also figure 4 for the procedure and results of this experiment.

Example 4

Detection of other Complex mutations

Diagnosis of clinical sample hemophilia A and other types of inversions by LD-PCR combined qPCR one-step method

It is now possible to demonstrate the use of a one-step method for detecting inversion of the 22 st intron in hemophilia a men, and in women carriers of inversion of the 22 st intron. Meanwhile, for the rare types of F8 Dup22 and F8 Del22 with inverted intron 22 of the F8 gene, LD-PCR combined Q-PCR one-step experiments theoretically detect the two rare types. The relative copy numbers of the templates that can be used for LD-PCR (P & Q primer pair and P &2F3R primer pair) are summarized in Table 6.

For the male example, the relative copy number of the template for P & Q was 1 and the relative copy number of the template for P &2F3R was 0 for the non-Inv 22 individuals, so that the amount of the template for M1 was greater than that for M2 after LD-PCR reaction, and confirmed by double qPCR quantification, as described above. In Inv22, since the relative copy number of P & Q template was 0 and that of P &2F3R was 1, the amount of M2 template was greater than that of M1 template after LD-PCR reaction, and confirmed by double qPCR quantification, as also described above. For the rarer Dup22, the template relative copy number of P & Q and P &2F3R were both 1, so after LD-PCR, the dual qPCR results for M1 and M2 would be between non-Inv 22 and Inv 22. For Del22, in the LD-PCR process, both P & Q and P &2F3R have no template and cannot be effectively amplified, the subsequent qPCR can only be used for amplification by using the original non-amplified M1 and M2 templates, and the absolute Ct values of M1 and M2 are higher.

TABLE 6

Example 5

Combination of LD-PCR and qPCR for detection of other chromosomal rearrangements

It will be appreciated by those skilled in the art that the combination of LD-PCR and qPCR of the present invention can also be used for the detection of other chromosomal rearrangements based on principles similar to those of the above examples. For example, more common chromosomal microdeletions (e.g., 1p36 deletion) can be detected, and more deletions can be addedSeehttps：//www.ncbi.nlm.nih.gov/pmc/articles/ PMC3681172/(ii) a And detecting fusion of the ALK gene with EML4 in lung cancer.

The detection process is shown in fig. 5 and 6.

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Claims

Use of primers of SEQ ID NO. 1-3 and SEQ ID NO. 4-7 and probes of SEQ ID NO. 8 and 9 for the preparation of a kit for the detection of chromosomal rearrangements by a method comprising the steps of:

a. obtaining sample DNA from a sample from a subject;

b. long-range PCR (LD-PCR) was performed on the sample DNA using primers of SEQ ID NO: 1-3;

c. quantitative PCR (qPCR) is carried out on the products obtained by LD-PCR by using the primers of SEQ ID NO. 4-7 and the probes of SEQ ID NO. 8 and 9; and

d. comparing the results of step c with the results of a normal control sample; and deducing from the result of the comparison whether a chromosomal rearrangement is present in the sample DNA,

wherein the chromosomal rearrangement is an inversion of intron 22 of the F8 gene.
Use of the primers of SEQ ID NO. 1-3 and SEQ ID NO. 10-13 and the probes of SEQ ID NO. 8 and 9 for the preparation of a kit for the detection of chromosomal rearrangements by a method comprising the steps of:

a. obtaining sample DNA from a sample from a subject;

b. LD-PCR and qPCR were performed on sample DNA in a single reaction vessel using primers of SEQ ID NO 1-3 and SEQ ID NO 10-13 and probes of SEQ ID NO 8 and 9; and

c. comparing the results of step b with the results of a normal control sample; and deducing from the result of the comparison whether a chromosomal rearrangement is present in the sample DNA,

wherein the chromosomal rearrangement is an inversion of intron 22 of the F8 gene.
3. The use of claim 1 or 2, wherein the sample from the subject is selected from the group consisting of blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, stool, and tissue samples.
4. The use of claim 1 or 2, wherein the sample from the subject is a bodily fluid.
5. A kit for detecting chromosomal rearrangements, comprising primers for performing LD-PCR on sample DNA from a subject and primers and probes for performing qPCR on the products of the LD-PCR, wherein the primers for performing LD-PCR and the primers and probes for performing qPCR are placed in separate containers, and,

wherein the primers used to perform LD-PCR are SEQ ID NO 1-3, and the primers and probes used to perform qPCR are SEQ ID NO 4-7 and SEQ ID NO 8-9, respectively.
6. A kit for detecting chromosomal rearrangements, comprising primers for performing LD-PCR on sample DNA from a subject and primers and probes for performing qPCR on the product of the LD-PCR, wherein the primers for performing LD-PCR and the primers and probes for performing qPCR are placed in the same container, and,

wherein the primers used to perform LD-PCR are SEQ ID NO 1-3, and the primers and probes used to perform qPCR are SEQ ID NO 10-13 and SEQ ID NO 8-9, respectively.
7. The primer is shown as SEQ ID NO 1-3 and SEQ ID NO 4-7 nucleotide sequences; or

Shown as the nucleotide sequences of SEQ ID NO. 1-3 and SEQ ID NO. 10-13.