CN116064775A

CN116064775A - Detection kit and detection method for TRECs, KRECs and SMN1

Info

Publication number: CN116064775A
Application number: CN202211402046.7A
Authority: CN
Inventors: 张巍; 田辉
Original assignee: Jiajian Guangzhou Bioengineering Technology Co ltd
Current assignee: Jiajian Guangzhou Bioengineering Technology Co ltd
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2023-05-05

Abstract

The invention relates to a detection kit and a detection method for TRECs, KRECs and SMN 1. By adopting the kit, the joint detection of SCID and SMA can be realized. Furthermore, based on the primer pairs and the probes, a multiplex fluorescence quantitative PCR method can be adopted, in a fluorescence quantitative amplification system, the specific amplification of TRECs, KRECs and SMN1 gene templates in a nucleic acid sample is realized, and the copy numbers of TRECs, KRECs and SMN1 in the peripheral blood cells in unit quantity are determined by further comparing CT value differences of target genes and internal reference genes, so that the rapid, efficient and sensitive detection of SCID and SMA is realized.

Description

Detection kit and detection method for TRECs, KRECs and SMN1

Technical Field

The invention relates to the field of gene detection, in particular to a detection kit and a detection method for TRECs, KRECs and SMN 1.

Background

Severe Combined Immunodeficiency Disease (SCID) is a rare group of congenital diseases caused by a variety of genetic factors characterized by a decrease in peripheral blood lymphocyte count (severe lymphopenia) and hypo-immunoglobulin emia, which results in humoral immunity and cellular immunity with a concomitant severe defect, which is often defined as primary T-cell deficiency. SCID occurs at a rate of about 1/10 th of a thousand newborns, but because SCID infants die often before definitive diagnosis, this figure can be severely underestimated. Without treatment, the mortality rate of SCID was 100%. At least 14 gene defects are associated with the phenotype typical of SCID, multiple sex linked stealth inheritance, and there are rare cases of autosomal stealth inheritance and sporadic disease. Depending on the gene deficiency, the lymphocyte phenotype of SCID patients can be divided into 4 classes: T-B+ NK-, T-B-NK-, T-B-NK+, and T-B+ NK+. Clinically, the infant has one or repeated infection in the first months of birth, and seriously threatens the life and the quality of life of the infant.

Mature T, B lymphocytes express specific T, B cell receptors (TCR and immunoglobulin Ig) on their surface, which are located on different chromosomes, and the locus is usually composed of three classes of genetic elements (variable region (V), variable region (D), and junction region (J)). During T, B cell maturation, the Recombination Signal Sequences (RSS) downstream of the V region, at both ends of the D region and upstream of the J region undergo (V-D-J) gene rearrangement by recombination events, and the sequences between the recombination elements are deleted from the chromosome by end-to-end formation of a circular structure, forming T cell receptor excision loops (TRECs) and B cell receptor excision loops (KRECs), respectively. For example: during the maturation process of TCR alpha chain coding genes, deleting (TCR) delta chain coding genes (forming delta Rec-phi J alpha TREC rings) through gene recombination between (V) alpha and (J) alpha; light chains of B cell receptors include both kappa and lambda chains, and B cells expressing lambda chains undergo a rearrangement of the lambda chain-encoding gene during maturation resulting in deletion of kappa chain-encoding gene ck and kappa enhancer sequences to form kappa recombination deletion loops (sjKRECs). These extrachromosomal circular DNA structures are stable, but cannot continue to divide, as a byproduct of T, B cell development and differentiation, and are gradually diluted as T, B cells divide. The copy number of TREC loops in the lymphocyte population can be used as a reliable indicator for determining the initial T lymphocyte function of thymus output in early neonatal development. Similarly, the number of copies of the KREC loop in a lymphocyte population can also be used as a biomarker for measuring B cell proliferation in peripheral lymphoid organs.

Chan and Puck first reported in 2005 the detection of T cell receptor excision loops using real-time quantitative PCR methods. EnLite Neonatal TREC Kit by PerkinElmer was approved by the FDA in 2014 in the united states, the first commercial kit for detecting severe infant syndrome immunodeficiency (SCID). The kit uses multiplex fluorescence quantitative PCR to obtain a few drops of blood from the heel of a neonate, and detects whether the T cell receptor excision loop DNA sequence (TREC DNA) in the neonate genome is reduced or deleted, which is a characteristic of a neonate with SCID. At present, neonatal SCID screening work has been conducted in a plurality of countries, however, clinical screening efficiency and sensitivity have yet to be improved. Therefore, it is of great importance to build a rapid, efficient, sensitive SCID screening technology system.

Spinal muscular atrophy (Spinal Muscular Atrophy, SMA) is an autosomal recessive neuromuscular disease that can have a great impact on the physical health and normal life of newborns, even on longevity, and is a major genetic cause of death in infants below 2 years of age. SMA early detection by neonatal screening is critical to pre-symptomatic treatment and to ensure that the patient obtains an optimal prognosis. SMA is generally identified by examination of serum creatine kinase, muscle biopsies, electromyography, and genetic testing. Type 4 is classified according to age of onset and clinical manifestation of SMA patients: type I (severe), type II (intermediate), type III (light) and type IV (adult). The causative gene of SMA is located in the region of chromosome five 5q11.2-5q13.2, where two highly homologous genes are designated motor neuron survival genes (survival motor neuron, SMN). Wherein SMN1 near the telomere is a pathogenic gene for SMA; SMN2 near the centrosome is not an SMA causative gene, but its copy number correlates with SMA clinical manifestation severity. 95% of SMA type I patients have symptoms caused by homozygous deletion mutations carrying exon 7 of the SMN1 gene. The target site of the exon of the SMN1 7 is detected by a real-time fluorescence quantitative PCR method, so that the quantification of the copy number of the normal SMN1 gene in human cells can be realized. If there are on average 2 copies of normal SMN1 gene per cell, it is completely normal, if there are on average only 1 normal SMN1 gene per cell, it is carrier, if there are no normal SMN1 genes in the cell, it is SMA patient. Compared with other detection methods, the qPCR detection method has the advantages of short time consumption, low equipment cost, high detection sensitivity and the like. Neonatal disease screening (neo-screening) refers to performing a dedicated examination of congenital and genetic diseases that are severely detrimental to neonatal health during neonatal period. The new screen is generally realized by detecting relevant indexes of the dry blood sheets of the filter paper through a laboratory method. Taking dry blood spot samples of heel blood made within 72 hours of neonate birth has been successfully used in a range of new screening programs including Phenylketonuria (PKU), congenital Hypothyroidism (CH), congenital Adrenocortical Hyperplasia (CAH), glucose-6-phosphatase deficiency (G6 PD). Because of the limited sample size of dried blood spots of newborns (only tens of microliters), the sensitivity requirement on the back-end detection technology is very high. With the increase of new screening projects and the increase of national screening rate, the use of the same sample to screen several genetic diseases simultaneously has become a development trend in this field.

Currently, there is no combined SCID and SMA detection product. In view of this, the present application is specifically proposed.

Disclosure of Invention

One of the purposes of the present application is to provide a detection kit comprising a primer pair and a probe for detecting TRECs, KRECs and SMN1 genes, with which combined detection of SCID and SMA can be achieved.

The purpose of the application can be achieved by the following technical scheme:

in a first aspect of the present application, there is provided a detection kit comprising a primer pair 1 and a probe 1 for detecting TRECs, a primer pair 2 and a probe 2 for detecting KRECs, and a primer pair 3 and a probe 3 for detecting SMN1 genes, or further comprising an internal reference primer pair and an internal reference probe for detecting internal reference genes;

wherein,,

the amplification product 1 of the primer pair 1 is the same as the amplification products of the primer pairs shown as SEQ ID No.2 and SEQ ID No.3, the probe 1 specifically recognizes the TREC loop recombination connection site on the amplification product 1,

the amplification product 2 of the primer pair 2 is identical with the amplification products of the primer pair shown as SEQ ID No.6 and SEQ ID No.7, the probe 2 specifically recognizes the KREC loop recombination connection site on the amplification product 2,

The amplification product 3 of the primer pair 3 is identical to the amplification products of the primer pairs shown in SEQ ID No.17 and SEQ ID No.19, and the probe 3 specifically recognizes the SNP locus of the SMN1 gene on the amplification product 3.

In some embodiments of the present application, the detection kit has one or more of the following technical features:

(1) The primer pair 1 is provided with an upstream primer 1 shown in SEQ ID No.2 and a downstream primer 1 shown in SEQ ID No. 3;

(2) The primer pair 2 is provided with an upstream primer 2 shown in SEQ ID No.6 and a downstream primer 2 shown in SEQ ID No. 7; and, a step of, in the first embodiment,

(3) The primer pair 3 has an upstream primer 3 shown in SEQ ID No.17 and a downstream primer 3 shown in SEQ ID No. 19.

In some embodiments of the present application, the 15 th and 23 rd nucleotides of the upstream primer 3 have LNA modifications, and the 13 rd nucleotide of the downstream primer 3 has LNA modifications.

(1) The probe 1 is shown as SEQ ID No. 4;

(2) The probe 2 is shown as SEQ ID No. 8; and, a step of, in the first embodiment,

(3) The probe 3 is shown as SEQ ID No. 24.

In some embodiments of the present application, the 12 th and 14 th nucleotides of probe 3 have LNA modifications.

In some embodiments of the present application, the reference gene is the RPP30 gene of a human.

In some embodiments of the present application, the detection probe and the reference probe are each independently labeled with a fluorescent reporter group and a fluorescent quenching group, and the labeled fluorescent reporter groups are different between different detection probes and between a detection probe and a reference probe.

In a second aspect of the present application, there is provided a method for detecting TRECs, KRECs and SMN1 genes for non-diagnostic purposes, the method comprising the steps of:

detecting a nucleic acid sample to be detected by using the detection kit provided in the first aspect, and judging whether the copy numbers of TRECs, KRECs and SMN1 genes in the nucleic acid sample to be detected are abnormal according to detection results.

In some embodiments of the present application, the annealing temperature for amplification is 58 ℃ -62 ℃.

In some embodiments of the present application, determining whether the copy numbers of the TRECs, KRECs, and SMN1 genes in the nucleic acid sample to be tested are abnormal based on the detection result comprises:

the Ct values corresponding to the probe 1, the probe 2 and the reference probe are respectively referred to as Ct _{Detection 1} 、Ct _{Detection 2} And Ct _{Internal reference} Calculate ΔCt ₁ ＝|Ct _{Detection 1} -Ct _{Internal reference} |，ΔCt ₂ ＝|Ct _{Detection 2} -Ct _{Internal reference} |，

ΔCt ₁ And ΔCt ₂ If the condition shown in the following (1) is satisfied, determining that the copy numbers of TRECs and KRECs in the nucleic acid sample to be tested are normal,

ΔCt ₁ and ΔCt ₂ If the condition shown in the following (2) is satisfied, determining that the copy numbers of TRECs and KRECs in the nucleic acid sample to be tested are abnormal,

ΔCt ₁ and ΔCt ₂ If the condition shown in (3) below is satisfied, determining that there is a possibility that there is an abnormality in the copy numbers of TRECs and KRECs in the nucleic acid sample to be tested, suggesting a re-detection,

ΔCt ₁ and ΔCt ₂ If the condition shown in the following (4) is satisfied, judging that the concentration of the nucleic acid sample to be detected is too low to judge;

(1)Ct _{internal reference} ≤27，ΔCt ₁ ≤10.0，ΔCt ₂ ≤10.0

(2)Ct _{Internal reference} ≤27，ΔCt ₁ > 12.5; alternatively, ct _{Internal reference} Ct shows "undetermined". Ltoreq.27; alternatively, ct _{Internal reference} Less than or equal to 27, although Ct values are shown, no obvious amplification curve is generated;

(3)Ct _{internal reference} More than or equal to 27, more than 10.0 and less than or equal to 12.5 of delta Ct 1; alternatively, ct _{Internal reference} ≤27，10.0＜ΔCt ₂ ；

(4)Ct _{Internal reference} ＞27，ΔCt ₁ > 10.0, with a clear amplification curve; alternatively, ct _{Internal reference} ＞27，ΔCt ₂ > 10.0, with a clear amplification curve; alternatively, ct _{Internal reference} Ct shows "undetermined" > 27; alternatively, there is no obvious amplification curve, although Ct values are shown;

the method comprises the steps of,

the Ct values corresponding to the probe 3 and the reference probe are respectively referred to as Ct values _{Detection 3} And Ct _{Internal reference} Calculate ΔCt ₃ ＝|Ct _{Detection 3} -Ct _{Internal reference} |，

ΔCt ₃ If the condition shown in the following (5) is satisfied, judging that the copy number of the SMN1 gene in the nucleic acid sample to be detected is normal,

ΔCt ₃ if the condition shown in the following (6) is satisfied, determining that the copy number of the SMN1 gene in the nucleic acid sample to be detected is abnormal,

ΔCt ₃ if the condition shown in the following (7) is satisfied, judging that the concentration of the nucleic acid sample to be detected is too low to judge;

(5)Ct _{internal reference} ≤30.0，ΔCt ₃ ≤8.0；

(6)Ct _{Internal reference} ≤30.0，ΔCt ₃ > 8.0; alternatively, ct _{Internal reference} Ct value shows "undetermined". Ltoreq.30.0; alternatively, ct _{Internal reference} Less than or equal to 30.0, although Ct values are shown, no obvious amplification curve is shown;

(7)Ct _{internal reference} ＞30.0，ΔCt ₃ > 8.0; alternatively, ct _{Internal reference} Ct value shows "undetermined". Gtoreq.30.0; alternatively, ct _{Internal reference} More than or equal to 30.0, the Ct value is shown, but no obvious amplification curve is generated.

Compared with the prior art, the beneficial effects of the application include:

the detection kit provided by the application comprises a primer pair and a probe for detecting TRECs, KRECs and SMN1 genes, and can realize joint detection of SCID and SMA. Furthermore, based on the primer pairs and the probes, a multiplex fluorescence quantitative PCR method can be adopted, and in a fluorescence quantitative amplification system, the specific amplification of TRECs, KRECs and SMN1 gene templates in a small amount of nucleic acid samples (for example, 2-3 mm dry blood spot DNA samples of newborns) is realized, and the copy numbers of TRECs, KRECs and SMN1 in a unit number of peripheral blood cells are determined by further comparing CT value differences (delta Ct values) of target genes and reference genes, so that the rapid, efficient and sensitive detection of SCID and SMA is realized. In particular, it is possible to achieve accurate quantitative detection of the SMN1 7 exon in the 0-3 range of SMN1 copy number (/ genome), thereby achieving high accuracy detection of SCID and SMA diseases by extracting a small number (2-3 mm diameter) of samples (e.g., dried blood spot samples) to obtain a nucleic acid sample as a template. The primer pair and the probe for detecting the No. 7 exon of the SMN1 gene are designed for 2 SNP loci on the No. 7 exon and the No. 7 intron between the SMN1 and the SMN2, and the primer and the probe are modified by using Locked Nucleic Acid (LNA) respectively, so that the primer pair and the probe have extremely high sensitivity and specificity, and can accurately quantify the 0, 1, 2 and 3 copies (/ genome) of the SMN1 7 exon.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort to a person skilled in the art.

FIG. 1 is a graph of ROC in example 7;

FIG. 2 is a graph of ROC for example 8.

Detailed Description

The present invention will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the invention and are not intended to limit the scope of the invention in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by those skilled in the art without departing from the spirit of the invention, and equivalents thereof fall within the scope of the present application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention, it being understood that the invention may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the invention.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or," "and/or," as used herein, includes any one of the two or more items listed in relation to each other, as well as any and all combinations of items listed in relation to each other, including any two or more of the items listed in relation to each other, or all combinations of items listed in relation to each other.

In this application, the terms "first," "second," "third," "fourth," "first," "second," "first segment," "second segment," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity or as implicitly indicating the importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.

In the present application, reference to a numerical range includes both endpoints of the numerical range unless otherwise indicated.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

The term "% identity" in the context of two or more nucleotide sequences or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is the entire length of the coding region relative to the sequences to be compared. For sequence comparison, typically one sequence is used as a reference sequence, and the test sequence is compared to that sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al, 1990, J Mol Biol 215:3,403-410;Altschul et al, 1997,Nucleic Acids Res25:17,3389-402).

The primer and probe modification may be performed by a known method. Modified versions of these primer and/or probe sequences can include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, one or more nucleotides to the 3' end, one or more nucleotides to the 5 'and 3' ends, adding tails, shortening the sequence, extending the sequence, shifting the sequence several bases upstream and downstream, or any combination thereof. Base modifications such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2 ' -deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2 ' -deoxyadenosine-5 ' -triphosphate, 2, 6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's) and include at least one mismatched base at one of the bases, or at least one of the bases is replaced with an RNA base, to effect, for example, an increase in nucleic acid interactions at the 3' end of the mutant-specific primer to increase Tm. The addition of double-stranded stable base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. The modified probe should retain the ability to distinguish between the mutation site to be detected and the wild-type site.

The term "probe" as used herein refers to any of a variety of signaling molecules that are indicative of amplification. For example, SYBR Green and other DNA binding dyes are detection probes. May be a sequence-based detection probe, such as a 5' nuclease probe. Some detection probes are known in the art, such as Taqman probes, stem-loop molecular beacons, MGB probes, scorpion probes, locked Nucleic Acid (LNA) probes, peptide Nucleic Acid (PNA) probes, and the like.

The term "kit" refers to any article of manufacture (e.g., package or container) comprising at least one device, which may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.

First aspect of the present application

The application provides a detection kit, which comprises a primer pair 1 and a probe 1 for detecting TRECs, a primer pair 2 and a probe 2 for detecting KRECs, and a primer pair 3 and a probe 3 for detecting SMN1 genes, or further comprises an internal reference primer pair and an internal reference probe for detecting internal reference genes;

wherein,,

Primer pair 1 of the present application may be as shown in SEQ ID No.2 and SEQ ID No.3, or may have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identity to the sequence of the primer pair and be identical to the amplification product of the primer pair.

Primer pair 2 of the present application may be as shown in SEQ ID No.6 and SEQ ID No.7, or may have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identity to the sequence of the primer pair and be identical to the amplification product of the primer pair.

Primer pair 3 of the present application may be as shown in SEQ ID nos. 17 and 19, or may have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identity to the sequence of the primer pair and be identical to the amplification product of the primer pair.

Optionally, the 15 th and 23 rd nucleotides of the upstream primer 3 have LNA modifications, and the 13 rd nucleotide of the downstream primer 3 has LNA modifications.

Probe 1 of the present application may be as shown in SEQ ID No.4, or may be another probe that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identity with the sequence of the probe and is capable of specifically recognizing the TREC loop recombination ligation site on amplification product 1.

Probe 2 of the present application may be as shown in SEQ ID No.8 or may be another probe having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identity to the sequence of the probe and capable of specifically recognizing the KREC loop recombination ligation site on amplification product 1.

Probe 3 of the present application, as shown in SEQ ID No.24, may also be other probes that have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identity to the sequence of the probe and are capable of specifically recognizing the SNP site of the SMN1 gene on the amplification product 3.

Optionally, the 12 th and 14 th nucleotides of the probe 3 have LNA modifications.

The selection of the reference gene is not particularly limited in the present application, and a gene satisfying the following conditions may be selected as the reference gene: the gene is a single copy gene and has no homologous pseudogene. Alternatively, the reference gene comprises an RPP30 gene.

In some embodiments of the present application, the detection probe and the reference probe are each independently labeled with a fluorescent reporter group and a fluorescent quenching group, and the labeled fluorescent reporter groups are different between different detection probes and between a detection probe and a reference probe. Alternatively, the fluorescent emitting groups of each probe are independently selected from any of AMCA, pacific Blue, atto425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aqua Phuor 593, texas Red, atto 590, cy5, quasar 670, cy5.5, and Cy5.5.

In one example, the detection kit further comprises an amplification buffer, a positive quality control, a negative quality control, or a combination thereof.

Alternatively, the amplification buffer may comprise dNTPs, mg ²⁺ UNG enzyme, DNA polymerase, and the like. Suitable polymerases for practicing the present application are well known in the art and can be obtained from a variety of sources. Thermostable DNA polymerases are available from a variety of commercial sources using methods well known to those skilled in the art. Preferred thermostable DNA polymerases can include, but are not limited to: taq DNA polymerase or a mutant, derivative or fragment thereof.

Alternatively, the components of the kit, such as the primer, probe, positive control, and negative control, may be stored in the kit in dry powder form. The positive control as well as the negative control may be present as plasmids. Alternatively, the components are preferably realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilization beads are generally understood to mean lyophilisates which are pressed into spheres after manufacture, after which the substance is usually present as a powder. Thus, the components required for a PCR batch, in particular the DNA polymerase, the nucleic acid components and the reaction buffer components, can be provided, for example, in lyophilized form. In this way, the PCR process can be started directly in a very user-friendly manner by adding the sample to be quantified and optionally other desired components. In particular, the provision of a lyophilized form is very advantageous for automated applications.

Second aspect of the present application

The application provides a detection method of TRECs, KRECs and SMN1 genes based on non-diagnostic purposes, which comprises the following steps:

In some embodiments of the present application, the annealing temperature for amplification is 58 ℃ -62 ℃ (e.g., 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62 ℃).

the Ct values corresponding to the probe 1, the probe 2 and the reference probe are respectively referred to as Ct _{Detection 1} 、Ct _{Detection 2} And Ct _{Internal reference} Calculate ΔCt ₁ ＝Ct _{Detection 1} -Ct _{Internal reference} ，ΔCt ₂ ＝Ct _{Detection 2} -Ct _{Internal reference} |，

ΔCt ₁ and ΔCt ₂ Satisfy the following (2)Judging that the copy numbers of TRECs and KRECs in the nucleic acid sample to be detected are abnormal under the conditions shown,

(1)Ct _{internal reference} ≤27，ΔCt ₁ ≤10.0，ΔCt ₂ ≤10.0

(4)Ct _{Internal reference} 27，ΔCt ₁ > 10.0, with a clear amplification curve; alternatively, ct _{Internal reference} ＞27，ΔCt ₂ > 10.0, with a clearly evident amplification curve; alternatively, ct _{Internal reference} Ct shows "undetermined" > 27; alternatively, there is no obvious amplification curve, although Ct values are shown;

the method comprises the steps of,

(5)Ct _{internal reference} ≤30.0，ΔCt ₃ ≤8.0；

The detection method of the embodiment of the application can be used for diagnosing/assisting in diagnosing spinal muscular atrophy (Spinal Muscular Atrophy, SMA) and SCID.

Samples for which the test kits, test methods of embodiments of the present application are applicable may be derived from, including but not limited to, cells, such as circulating blood, cultured cells, tumor cells. Sources of samples such as one or more of pharyngeal swabs, nasal swabs, sputum, respiratory aspirates, bronchial lavages, alveolar lavages, conjunctival swabs, saliva samples, stool specimens, anticoagulants, and serum specimens. The DNA may be DNA in the genome or in a plasmid or other vector. The application is used for detecting the SNP locus of the SMN1 gene in the genome, and can be primate (especially human) or any other animal known or unknown with the SNP locus of the SMN1 gene. In some embodiments, the template sequence or nucleic acid sample may be gDNA. In other embodiments, the source of the template sequence or nucleic acid sample may be any type of tissue, including, for example, formalin-fixed paraffin-embedded tissue samples.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present invention, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Embodiments mainly comprise the following contents:

the sequences of the primer pairs and probes for detecting TRECs and KRECs were determined. According to the structural characteristics of TRECs and KRECs, the upstream primer and the downstream primer for detecting TRECs and KRECs are respectively designed on two sides of TRECs and KRECs by using primer and probe design software. In addition, a Taqman probe sequence is designed near the recombination site of TRECs and KRECs, FAM fluorescent groups and BHQ1 quenching groups are respectively added at the 5 'end and the 3' end of the TREC probe, and FAM fluorescent groups and BHQ1 quenching groups are respectively added at the 5 'end and the 3' end of the KREC probe.

Further, the sequences of the primer pair and probe for SMN1 gene detection were determined. Designing forward and reverse primers of SMN1 aiming at SNP loci of the SMN1/2 in exon7 and intron 7 by using primer design software, wherein the forward primer comprises the SNP locus of exon7, the reverse primer comprises the SNP locus of the intron 7, and LNA is added into a primer sequence to modify and improve the Tm value and amplification specificity of the primers; and a section of SMN1/2 public sequence is selected by utilizing probe design software to design an SMN1 probe, and CY5 fluorescent groups and BHQ1 quenching groups are respectively added at the 5 'end and the 3' end of the SMN1 probe.

Further, the sequences of the primer pair and probe for detecting the reference gene were determined. In order to conveniently quantify the copy number of the target gene, a single copy gene RPP30 is selected as a reference gene, and primer pairs and probes are designed in a highly conserved region of the RPP30 by using primer and probe design software. And, VIC fluorescent groups and MGB quenching groups are added to the 5 'end and the 3' end of the reference gene probe, respectively.

The application selects the primer pair and the probe for the biological synthesis design of Shanghai workers. Further, 2X Taqman Fast Advanced Master Mix (thermosusher, cat. # 4444557) was selected as an enzyme premix for the reaction, which premix contains a reaction buffer, a ROX reference dye, and the like. Wherein the reaction buffer solution contains dNTP, mg2+, UNG enzyme and DNA polymerase.

The application can select the ABI7500 fluorescent quantitative PCR instrument as the model of the reaction system test.

The parameters of the designed primer pair, the probe and the reaction system are tested, optimized and confirmed.

First, the amplification of the blank template by the primer and probe combination was examined in a singleplex system, and the specificity of the reaction system, i.e., the blank template was confirmed to have no nonspecific amplification signal.

Further, a series of concentration gradient (copy number) quadruple (PUC 57-SMN1-TREC-KREC-RPP30, PUC57-SMN2-TREC-KREC-RPP 30) and singleplex (PUC 57-RPP 30) plasmids were prepared using gradient dilutions of plasmid templates of known copy numbers for negative and positive quality control of the reaction system.

Furthermore, the optimal annealing temperature and the amplification efficiency of each primer pair/probe combination are tested by utilizing a TREC, KREC, SMN and RPP30 single reaction system and plasmid templates with different copy numbers, and a primer pair/probe combination with similar amplification efficiency and a Ct value amplified by the same copy number template is selected to construct a multiple system.

Furthermore, the forward and reverse primer and probe sequences of the SMN1 are systematically screened and optimized in a single, double and quadruple real-time fluorescent quantitative reaction system, and the SMN1 primer and probe combination which has good amplification effect on the SMN1 template and has no amplification signal on the SMN2 template is selected for subsequent detection.

Furthermore, the concentration combination of TREC, KREC, SMN and RPP30 four primer pairs/probes in the quadruple real-time fluorescence quantitative reaction system is optimized, and the concentration combination with consistent amplification efficiency and Ct value for four templates of a tandem plasmid (PUC 57-SMN1-TREC-KREC-RPP 30) is selected for primer and probe concentrations of the quadruple system.

Furthermore, the performance of the quadruple fluorescence quantitative system is researched and verified by using clinical nucleic acid samples with different concentrations, wherein the performance comprises the detection linear range of the reaction system in a quadruple fluorescence channel, the positive judgment value confirmation of SCID and SMA, the detection LOD confirmation of TREC, KREC and SMN1 templates, and the detection precision confirmation of the system and the middle, low-concentration negative and positive samples.

The linear range of the quadruple system TREC (FAM channel) was tested with a known copy number of TREC plasmid standard, which indicated that: the linear range of TREC detection is 30-1000 copies/reaction, ct value: 30-35.0; the linear range of the quadruple system KREC (NED channel) was tested with known copy number of KREC plasmid standards, which indicated that: the linear range of KREC detection is 30-1000 copies, ct:29.4-34.5; after gradient dilution of the genomic DNA of the dried blood spots with known RPP30 copy number, the linear range of RPP30 (VIC channel) was used as a template for the quadruple system of the template test, and the results showed that: the linear range of RPP30 detection is 44-5724 copies, ct:26.8-34.1; after gradient dilution of known SMN1 copy number of dry blood spot genomic DNA, the linear range of the quadruple system SMN1 (CY 5 channel) was used as a template test, and the results showed that: the linear range of SMN1 detection is 44-5724 copies, ct:26.6-34.1.

Sample populations for which SCID mutation has been confirmed by second generation sequencing are detected using a SCID/SMA quadruple detection system (fluorescence PCR method), and the difference ΔCt between TREC Ct value and internal reference RPP30 Ct value is analyzed by ROC curve to determine the positive judgment value (Ct _RPP30 ≤27.0,Ct _TREC -Ct _RPP30 >12.5). According to the formulated SCID positive judgment value, a quadruple detection system is used for further detecting samples with related gene mutation confirmed by second generation sequencing, and the detection result is consistent with the second generation sequencing result, so that the method for setting the SCID positive judgment value is reasonable.

Sample populations for which SMN1 mutation had been confirmed by second generation sequencing were examined using a SCID/SMA quadruple detection system (fluorescent PCR method), and the difference ΔCt between the SMN1 Ct value and the internal reference RPP30 Ct value was analyzed by ROC curve to determine the positive determination value (Ct) of the quadruple detection system for the SMN1 gene _RPP30 ≤30.0,Ct _SMN1 -Ct _RPP30 >8.0). Further detecting by using a quadruple fluorescence quantitative detection system according to the formulated SMA positive determination valueAnd (3) testing samples with related SMN1 gene mutation conditions confirmed by second-generation sequencing, wherein the detection result is consistent with the second-generation sequencing result, so that the method for setting the SMA positive judgment value is reasonable.

And (3) diluting the dried blood spot DNA sample to a detection limit concentration by using an enzyme-free water gradient, using the dried blood spot DNA sample as a template, researching the detection Limit (LOD) of TREC, KREC, SMN on a quadruple system, taking a DNA template with low copy number, repeatedly detecting for 20 times, and calculating the detection rate of 20 detection results. When the detection rate of the 20 detection results is more than or equal to 95 percent (the fluorescence channel to be detected has detection signals, and the Ct value is less than 40.0), and the Ct value CV is less than or equal to 5 percent, the lowest concentration meeting the condition is taken as the detection limit. The LOD test result shows that the lowest detection limits of TREC, KREC, SMN1 are respectively: 8 copies/reaction, 9 copies/reaction, 51 copies/reaction.

To test the detection precision of the quadruple system, the same tester was used to test SCID negative samples of the same concentration (medium concentration (Ct _RPP30 =25.0), low concentration (Ct _RPP30 =27.0)) and SCID positive samples (medium concentration (Ct) _RPP30 =25.0)), and SMA negative samples of the same concentration (medium concentration (Ct) _RPP30 =25.0), low concentration (Ct _RPP30 =30.0)) and SMA positive samples (medium concentration (Ct _RPP30 =25.0)) was performed consecutively for 10 days, 10 replicates per sample. The detection result shows that: the negative/positive coincidence rate of the SCID/SMA negative and positive samples is 100%, and the variation coefficient (cv%) of Ct value of the same sample detection<5%. The quadruple detection reagent has good precision.

The specific operation is as follows:

example 1: screening of primers and probes

Step 1: design of TREC primer probes

Searching for the sequence of the TREC receptor loop near the recombination junction: and (3) searching sequence information of TREC receptor rings (delta Rec/phi J alpha) formed by TCR alpha on chromosome 14 in the recombination process on NCBI at the upper and lower streams of recombination splicing sites, intercepting the 200bp sequences at the upper and lower streams of the recombination splicing sites, and carrying out primer probe design, wherein underlined nucleotides are the recombination splicing sites of the TREC rings.

CTTATTCATTGTCTTCATCCCTGAAATACACTCTGCTCTCTCCTATCTCTGCTCTGAAAGGCAGAAAGAGGGCAGCCCTCTCCAAGGCAAAATGGGGCTCCTGTGGGGAACAGAGGGGTGCCTCTGTCAACAAAGGTGATGCCACATCCCTTTCAACCATGCTGACACCTCTGGTTTTTGTAAAGGTGCCCACTCCTGTGCACGGTGATGCATAGGCACCTGCACCCCGTGCCTAAACCCTGCAGCTGGCACGGGCCCTGTCTGCTCTTCATTCACCGTTCTCACGAGTTGCAATAAGTTCAGCCCTCCATGTCACACT(SEQ ID No.1)

Under the assistance of Primer and probe design software oligo 7 and Primer Premier 5.0 software, an upstream Primer is designed at the 5 'end of a TREC recombination connection site, a downstream Primer is designed at the 3' end of the recombination connection site, a probe sequence is designed near the recombination connection site, and the TREC specific primers and probe sequences are shown in Table 1.

TABLE 1

Step 2: design of KREC primer probe

Search for sequences of kappa receptor recombination deletion loop (sjKREC) near the recombination junction: the sequence information of a kappa receptor recombination deletion loop (sjKREC) on chromosome 2 near a recombination splice site (Ekappa-Ckappa) is searched on NCBI, and the sequences of 150 bp and 200bp above and below the recombination junction of the sjKREC loop are intercepted for primer probe design, and the underlined two nucleotides are the recombination junction site of the KREC loop.

ATTAAATAGCACTGAAAAAAAAAAAAGCTTTAAATTATTTACAATCCCCTAATGGAAATTTTCACTAATGAGATATCATAATGAATGTGAATTTTATTTCTGAAATCTCTAATAAATCAGTCTTCTCCCTGGTTTTCCCAGCTCAGCGCCCATTACGTTTCTGTTCTCTTTCCCTTAGTGGCATTATTTGTATCACTGTgcTAGGGCTCCCACAGTGTGCGCTGCCAACCTGCTGCCCGTGCAGAAACTCTAGGGTAAGAGCTGGCTCCTGGAGTCCCACCCAGGCTGCGTGTCCCTCACAGTCTGCTCTGTGTCTATGTGTGTGTGTTGGGGGGATATTATTGGACAATTCAAGGGAGGCTCAGTGAGGAGGAAGGAAAATTTCATGTAATTCTTTCTG(SEQ ID No.5)

With the aid of Primer and probe design software oligo 7 and Primer Premier 5.0 software, an upstream Primer was designed at the 5 'end of the KREC cleavage site, a downstream Primer was designed at the 3' end of the cleavage site, and probe sequences were designed at the cleavage site, as shown in Table 2.

TABLE 2

Step 3: design of SMN1 primer probes

The SMN1 complete gene sequence on chromosome 5 is searched on NCBI, and the SMN1 gene sequence containing SMN1 intron 6, exon 7 and intron 7 is selected as a template, wherein underlined nucleotides are the SMN1 gene specific nucleotide sequences.

ATAAAGCTATCTATATATAGCTATCTATGTCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTACAGGGTTTCAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAAAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTATGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATT(SEQ ID No.9)

The primers and probes were designed on the selected SMN1 sequences with the aid of Primer and probe design software oligo 7 and Primer Premier 5.0 software. Wherein the upstream primer of SMN1 covers the SMN1 specific site of exon 7; the downstream primer covers the SMN1 specific site on intron 7; the probe of SMN1 is a common sequence of SMN1 and SMN 2. In order to improve the specificity of the forward and reverse primer pair SMN1 template recognition and the amplification efficiency of the primer probe in a qPCR reaction system, specific nucleic acids on the primers and the probes are modified by LNA (locked nucleic acid), and the primers and the probes of the SMN1 are shown in Table 3:

table 3: primer and probe sequences of SMN1

Step 4: internal reference primer and probe design and selection

The gene RPP30 is selected as an internal reference gene, which is a single copy gene and has no homologous pseudogene. The highly conserved sequences were selected and primers and probes were designed with the aid of Primer and probe design software oligo 7 and Primer Premier 5.0 software, see table 4.

ATGGGACTTCAGCATGGCGGTGTTTGCAGATTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTCACCGTGAGTTGCCCCGGCTTCGCGCCTGGCCAACCTCATGCCACCCAGACCATCGGGCCACACTCCGGAGTAACTATTTCCTGATGGGTCTCGGTCAGGTCTCCCAGA(SEQ ID No.25)

TABLE 4 Table 4

Example 2: TREC/KREC/RPP30 primer and probe test

Step 1: testing of primers and probes for blank control

Blank controls were tested using 3 pairs of primer probes (TREC, KREC, RPP) in tables 1, 2 and 4 and SMN1 primers and probe combinations in table 3, respectively, and the test results are shown in table 5.

Table 5: detection results of primer probe on blank control

Sample of	TREC-F/R/P	KREC-F/R/P	RPP30-F/R/P	SMN1-F(1-8)/R(1-2)/P(1-6)
					NTC	Undet.	Undet.	Undet.	Undet.
NTC	Undet.	Undet.	Undet.	Undet.
					NTC	Undet.	Undet.	Undet.	Undet.

Step 2: preparation of known copy number plasmid samples

The PUC57-TREC-KREC-RPP30-SMN1 tandem plasmid, which contains TREC, KREC, RPP, SMN1 target sequence and TREC, KREC, SMN, RPP30 recognition sites for four pairs of primer probes, was used as template for primer probe testing and negative quality control for the quadruple system.

GAATTCAGGCACGGGGTGCAGGTGCCTATGCATCACCGTGCACAGGAGTGGGCACCTTTACAAAAACCAGAGGTGTCAGCATGGTTGAAAGGGATGTGGCATCACCTTTGTTGACAGAGGCAGGTACCTCGCGAATGCATCTAGACTGTTCTCTTTCCCTTAGTGGCATTATTTGTATCACTGTGCACAGTGTGCGCTGCCAACCTGCTGCCCGTGCAGAAACTCTAGGGTAAGAGCTGGCTCCTGGAGTCCCACGGATCCCGGGCCCATAAAGCTATCTATATATAGCTATCTATGTCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTACAGGGTTTCAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAAAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGTCGACTTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTCAAGCTT(SEQ ID No.29)

A PUC57-TREC-KREC-RPP30-SMN2 tandem plasmid containing the recognition sites of the TREC, KREC, RPP, SMN2 and TREC, KREC, RPP three pairs of primer probes was used as a control for the SMN1 primer probe test.

GAATTCAGGCACGGGGTGCAGGTGCCTATGCATCACCGTGCACAGGAGTGGGCACCTTTACAAAAACCAGAGGTGTCAGCATGGTTGAAAGGGATGTGGCATCACCTTTGTTGACAGAGGCAGGTACCTCGCGAATGCATCTAGACTGTTCTCTTTCCCTTAGTGGCATTATTTGTATCACTGTGCACAGTGTGCGCTGCCAACCTGCTGCCCGTGCAGAAACTCTAGGGTAAGAGCTGGCTCCTGGAGTCCCACGGATCCCGGGCCCATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTACAGGGTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGTCGACTTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTCAAGCTT(SEQ ID No.30)

Based on the size of the plasmid (3041 bp) and the molecular weight of the base (1.096X10) ^-21 g/bp) calculate mass of individual plasmid molecules: m is m _p ＝(3041bp)×(1.096×10 ^-21 g/bp)＝3.33×10 ^-18 g, mass of specific copy number was calculated from molecular weight of plasmid as shown in Table 6:

table 6: quality of quadruple tandem plasmid with gradient dilution copy number

The concentration of plasmid (g/. Mu.l) was calculated from the mass of plasmid and the volume of plasmid template added per reaction tube (5. Mu.l), as shown in Table 7:

TABLE 7

The plasmid templates were subjected to gradient dilution to specific concentrations (g/. Mu.l) as shown in Table 8:

TABLE 8

The PUC57-RPP30 plasmid, which contains the RPP30 sequence and the recognition site of the internal reference gene primer probe, was used as positive quality control for the quadruple system.

TTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTC(SEQ ID No.31)

Based on the size of the plasmid (2710 bp) and the molecular weight of the base (1.096X10) ^-21 g/bp) calculate mass of individual plasmid molecules: m is m _p ＝(2710bp)×(1.096×10 ^-21 g/bp)＝2.97×10 ^-18 g, mass of specific copy number was calculated from molecular weight of plasmid as shown in Table 9:

table 9: quality of positive quality control plasmid of gradient dilution copy number

The concentration of plasmid (g/. Mu.l) was calculated from the mass of plasmid and the volume of plasmid template added per reaction tube (5. Mu.l), as shown in Table 10:

table 10

Positive quality control plasmid templates were subjected to gradient dilution to specific concentrations (g/. Mu.l) as shown in Table 11:

TABLE 11

Step 3: test TREC, KREC, RPP amplification of primer probes to plasmid templates in a Single-reaction System

Results: comparing the amplification efficiency and fitting value of the plasmid templates in each single system at different annealing temperatures, wherein the annealing temperature value is 60 ℃, and the single system has the optimal amplification effect, so that the annealing temperature of the subsequent reaction system is selected to be 60 ℃.

Table 12: configuration of a single reaction System

Table 13: single qPCR reaction conditions

Results of plasmid template amplification in gradient dilutions in three single reaction systems TREC, KREC, and RPP 30. See tables 14, 15, 16, 17 and 18.

Table 14: test results of single reaction system on plasmid templates at different annealing temperatures

Table 15: test results of single reaction system on plasmid templates at different annealing temperatures

Table 16: test results of single reaction system on plasmid templates at different annealing temperatures

Table 17: test results of single reaction system on plasmid templates at different annealing temperatures

Table 18: test results of single reaction system on plasmid templates at different annealing temperatures

Example 3: TREC/KREC/RPP30 triple system primer probe concentration optimization

Step 1, configuring 6 different primer probe combinations

Table 19: concentration combinations of 6 primer probes tested in TREC/KREC/RPP30 triple system

The amplification reaction was performed based on the reaction system shown in Table 12 and the reaction conditions shown in Table 13 using the primer probe combinations shown in Table 19, and the primer probe combinations shown in Table 19 correspond to systems 1a to 6a in Table 20.

Step 2: investigation of the results of amplification of the gradient diluted plasmid templates by 6 different primer probe concentration combinations

Acceptance criteria: amplification efficiency of plasmid templates by three fluorescent channels: 95% or less and 105% or less of eff, R ² >0.999；ΔCt(Ct _TREC/KREC -Ct _RPP ) Absolute value of (2)<1.0。

The results of the test are shown in Table 20, and the primer probe combination 6a has the best amplification effect, and this concentration combination is used for the establishment of a triplex system.

Table 20: amplification results of gradient diluted plasmid templates (x 10-fold) under different primer probe combinations

Example 4: investigation of the linear Range of TREC/KREC/RPP30 triple System amplification

Step 1: testing of linear range of TREC (FAM channel) using TREC plasmid of known copy number

Table 21: gradient dilution of TREC plasmids of known copy number

Initial concentration (copy number/5. Mu.l)	Plasmid DNA volume (μl)	TE(μl)	Final volume (μl)	Final concentration (copy number/5. Mu.l)
					1000	20	0	20	1000
500	10	10	20	500
					250	10	10	20	250
125	10	10	20	125
					63	10	10	20	63
31	10	10	20	31
					16	10	10	20	8

Table 22: testing of linear ranges of FAM channels in a triple system with TREC plasmid

Step 2: testing of the linear Range of KREC (NED channel) Using known copy number of KREC plasmids

Table 23: gradient dilution of KREC plasmid standards

Table 24: testing the linear Range of NED channels with KREC standards in a triple System

Step 3: testing of the Linear Range of reference genes (RPP 30 (VIC channel)) Using gradient diluted Dry blood spot DNA

Table 25: gradient dilution of genomic DNA from dried blood spots with known RPP30 copy number

Initial concentration (copy number/5. Mu.l)	Plasmid DNA volume (μl)	TE(μl)	Final volume (μl)	Final concentration (copy number/5. Mu.l)
					5624	20	0	20	5624
5624	10	10	20	2812
					2812	10	10	20	1406
1406	10	10	20	703
					703	10	10	20	351
351	10	10	20	176
					176	10	10	20	88
88	20	0	20	44

Table 26: linear range of VIC channels in a triple system using gradient diluted dry blood spot DNA samples

Example 5: screening of SMN1 primers and probes

Step 1: the amplification of the same dried blood spot DNA (5.7 ng/. Mu.L) template by 6 different combinations of SMN1 primer probe singleplex systems was examined and compared with the Ct value of the RPP30 singleplex amplification system as a control

Inspection standard: according to the delta Ct value (Ct _SMN1 -Ct _RPP ) The SMN1 primer and probe combination with the highest amplification efficiency (minimum delta Ct value) is selected.

Results: the SMN1 primer probe combination 1b-6b and the formula thereof are shown in tables 27 and 28, the amplification result of the single system on the dried blood spot DNA sample is shown in table 29, wherein the combination 2b has the optimal Ct value in the single system, and the SMN1 primer and the probe in the combination are added with the modification of the locked nucleic acid, so that the qPCR reaction effect is improved, and the SMN1-R2 and the SMN1-P2 in the combination are selected, so that the SMN1-F is continuously optimized on the basis, namely the follow-up single system 7b-12b is optimized.

Table 27: primer and probe combinations of SMN1 single-weight system 1b-6b

Composition of the components

Combination 1b

Combination 2b

Combination 3b

Combination 4b

Combination 5b

Combination 6b

Primer F

SMN1-F1

SMN1-F2

SMN1-F1

SMN1-F2

Primer R

SMN1-R1

SMN1-R2

SMN1-R1

Probe with a probe tip

SMN1-P1

SMN1-P2

SMN1-P1

SMN1-P2

SMN1-P1

SMN1-P2

Table 28: reaction liquid formula of SMN1 single-weight system 1b-6b and RPP30 single-weight system

Component (A)	SMN reaction system	RPP30 reaction system
			2×Taqman Fast Advanced Master Mix	10μl/reaction	10μl/reaction
F(10μM)	0.4μl/reaction	0.4μl/reaction
			R(10μM)	0.4μl/reaction	0.4μl/reaction
P(10μM)	0.2μl/reaction	0.2μl/reaction
			H2O	4μl/reaction	4μl/reaction

Table 29: amplification results of dried blood spot DNA samples by the combination of the singleplex systems 1b-6b

Step 2: amplification of the same dry blood spot DNA (5.7 ng/uL) template by the single-strand system combination 7-12 was examined and compared with the Ct value of the control RPP30 single-strand amplification system, wherein the primer probe combination is shown in Table 30, and the reaction solution formulation of the single-strand system and the reaction solution formulation of RPP30 are shown in Table 31.

Inspection standard: according to the delta Ct value (Ct _SMN1 -Ct _RPP ) And selecting SMN1 primer and probe combinations with minimum delta Ct value.

Results: the amplification results of SMN1 and RPP30 singleplex systems are shown in Table 32, and among the primer probe combinations 7b-12b, singleplex systems 8b, 10b, 11b, 12b have Ct values that are relatively minimal and close to the RPP30 system, so that the four combined primer probes were selected for further investigation in the SMN1/RPP30 duplex qPCR system.

Table 30: primer and probe combination of SMN1 single-weight system 7b-12b

Composition of the components

Combination 7b

Combination 8b

Combination 9b

Combination 10b

Combination 11b

Combination 12b

Primer F

SMN1-F3

SMN1-F4

SMN1-F5

SMN1-F6

SMN1-F7

SMN1-F8

Primer R

SMN1-R2

Probe with a probe tip

SMN1-P2

Table 31: reaction liquid formula of SMN1 single-weight system 7b-12b and RPP30 single-weight system

Component (A)	SMN reaction system	RPP30 reaction system
			2×Taqman Fast Advanced Master Mix	10μl/reaction	10μl/reaction
F(10μM)	0.4μl/reaction	0.4μl/reaction
			R(10μM)	0.4μl/reaction	0.4μl/reaction
P(10μM)	0.2μl/reaction	0.2μl/reaction
			H ₂ O	4μl/reaction	4μl/reaction

Table 32: amplification results of dried blood spot DNA samples by SMN1 single system 7b-12b and RPP30 single system

Step 3: testing of SMN1/RPP30 Dual System

The SMN1 primer/probe combinations 2b, 8b, 10b, 11b and 12b and the primer probe of the RPP30 are selected to construct a double detection system, and the combinations are shown in a table 33; the concentrations of primers and probes in the duplex system are shown in Table 34, and the amplification effect of 5 duplex systems on the plasmid templates of PUC57-TREC-KREC-RPP30-SMN1 and PUC57-TREC-KREC-RPP30-SMN2, and the known SMA positive (SMN 1:0 copy, SMN2:3 copy) dry blood spot DNA was examined.

Inspection standard: a primer probe combination with high amplification efficiency on the SMN1 and RPP30 templates and no amplification signal on the SMN2 template is selected for the subsequent testing of a quadruple system.

Results: amplification efficiency (eff%) and Ct of the pUC57-TREC-KREC-RPP30-SMN1 (Table 35), the pUC57-TREC-KREC-RPP30-SMN2 plasmid template (Table 36) according to the double System _SMN1 -Ct _RPP The value, and the amplification result of the dry blood spot DNA (Table 37), the SMN1 primer and the probe of the combination 5c have high amplification efficiency and amplification specificity at the same time, so the combination is selected for constructing a quadruple system, and the next optimization test is carried out.

Table 33: SMN1 primer and probe used by combination of SMN1/RPP30 double systems

Table 34: formula of double-system primer probe

Component (A)	SMN/RPP30 reaction system
		2×Taqman Fast Advanced Master Mix	10μl/reaction
SMN1-F(10μM)	0.4μl/reaction
		SMN1-R(10μM)	0.4μl/reaction
SMN1-P(10μM)	0.2μl/reaction
		RPP3-F(10μM)	0.15μl/reaction
RPP3-R(10μM)	0.15μl/reaction
		RPP3-R(10μM)	0.1μl/reaction
H ₂ O	3.6μl/reaction

Table 35: amplification results of the double SMN1/RPP30 System on PUC57-TREC-KREC-RPP30-SMN1

Table 36: amplification results of the double SMN1/RPP30 System on the PUC57-TREC-KREC-RPP30-SMN2 plasmid

Table 37: detection results of double SMN1/RPP30 System on dried blood spot DNA sample (SMN 1:0 copy, SMN2:3 copy)

SMN1/RPP30 double system	Combination 1c	Combination 2c	Combination 3c	Combination 4c	Combination 5c
						Ct _SMN1	NaN	25.45	30.40	36.50	NaN
Ct _RPP30	25.50	25.40	25.48	25.58	25.35
						SMN1 copy number/reaction	/	13081	474	9	/
RPP30 copy/reaction	12651	13797	13331	11766	13984
						SMN1 detection specificity	High height	Low and low	Low and low	Low and low	High height

Step 4: quadruple detection system

And (3) selecting the SMN1 primer and probe of the combination 5c to construct a TREC/KREC/RPP30/SMN1 quadruple detection system, and examining the amplification effect of the quadruple system of 12 different primer and probe concentration combinations (Table 38) on the PUC57-TREC-KREC-RPP30-SMN1 plasmid template.

Inspection standard: selecting the optimal amplification efficiency (eff%) (the amplification efficiency of the four fluorescent channels is between 90 and 110 percent, R ² ＞0.999，(Ct _FAM/NED/CY5 -Ct _VIC ) Absolute value less than or equal to 1.0).

Results: the TREC/KREC/RPP30/SMN1 quadruple detection system concentration combination 10d has the optimal amplification effect on the plasmid template.

Table 38: concentration combinations 1d-12d (μl/reaction) of TREC/KREC/RPP30/SMN1 quadruple detection system

Table 39: concentration combinations 1d to 3d amplification results on the plasmid of the PUC57-TREC-KREC-RPP30-SMN1 quadruple system

Table 40: concentration combinations 4d to 6d amplification results of the pUC57-TREC-KREC-RPP30-SMN1 quadruple system plasmid

Table 41: concentration combinations 7d to 9d amplification results on the plasmid of the PUC57-TREC-KREC-RPP30-SMN1 quadruple system

Table 42: amplification results of the plasmid of the PUC57-TREC-KREC-RPP30-SMN1 quadruple System with concentration combination 10d to concentration combination 12d

Step 5: the primer and probe sequences of TREC, KREC, RPP and the forward and reverse primer sequences of SMN1 in the quadruple system combination 5d were selected, and on the basis of the selection, the SMN1 probes were further screened, and the effect of 4 different SMN1 probe sequences (Table 43) on the amplification effect of the quadruple system was examined.

Screening criteria: selecting a plasmid template of PUC57-TREC-KREC-RPP30-SMN1 and a SMA negative dry blood spot DNA sample (SMN 1:1 copy/genome) with good amplification efficiency (eff%) (the amplification efficiency of four fluorescent channels is between 90% and 110%, R ² ＞0.999，(Ct _FAM/NED/CY5 -Ct _VIC ) Absolute value is less than or equal to 1.0), the amplification specificity is strong (no amplification signal to the PUC57-TREC-KREC-RPP30-SMN2 plasmid and the SMA positive dry blood spot DNA template (SMN 1: 0 copy/genome).

Results: four SMN1 probes had the same sequence but different numbers of LNA modifications, and the four probes were compared for their amplification effects on the PUC57-TREC-KREC-RPP30-SMN1, PUC57-TREC-KREC-RPP30-SMN2, and the dried blood spot DNA template in a quadruple system (tables 44-46), indicating that the quadruple system comprising SMN1-P2 had the best amplification effect (amplification efficiency & specificity).

Table 43: primer probe combination of quadruple system 5d, 13d, 14d and 15d

Quadruple system	Combination 5d	Combination 13d	Combination 14d	Combination 15d
					SMN1-F	SMN1-F8	SMN1-F8	SMN1-F8	SMN1-F8
SMN1-R2	SMN1-R2	SMN1-R2	SMN1-R2	SMN1-R2
					SMN1-P	SMN1-P2	SMN1-P3	SMN1-P4	SMN1-P5
RPP30-F	RPP30-F	RPP30-F	RPP30-F	RPP30-F
					RPP30-R	RPP30-R	RPP30-R	RPP30-R	RPP30-R
RPP30-P	RPP30-P	RPP30-P	RPP30-P	RPP30-P
					TREC-F	TREC-F	TREC-F	TREC-F	TREC-F
TREC-R	TREC-R	TREC-R	TREC-R	TREC-R
					TREC-P	TREC-P	TREC-P	TREC-P	TREC-P
KREC-F	KREC-F	KREC-F	KREC-F	KREC-F
					KREC-R	KREC-R	KREC-R	KREC-R	KREC-R
KREC-P	KREC-P	KREC-P	KREC-P	KREC-P

Table 44: amplification of the pUC57-TREC-KREC-RPP30-SMN1 plasmid template by the quadruple System

Table 45: amplification of the pUC57-TREC-KREC-RPP30-SMN2 plasmid template by the quadruple System

Table 46: amplification of SMA negative & SMA positive dried blood spot DNA by quadruple system

Example 6: research on amplification efficiency of quadruple system and linear range of each fluorescent channel

Step 1: test of the linear range of quadruple TREC (FAM channel) with TREC plasmid of known copy number A gradient dilution of TREC plasmid of known copy number was performed according to the method of Table 21, and TREC templates of different copy numbers were tested.

Table 47: testing the linear range of FAM channels in a quadruple system with TREC standards

Step 2: testing of the Linear Range of KREC (NED channel) Using KREC plasmid standards

Known copy numbers KREC were subjected to gradient dilution according to the method of table 23, and KREC templates of different copy numbers were tested.

Table 48: testing the linear range of NED channels in a quadruple system with known copy number KREC templates

Step 3: testing of the Linear Range of RPP30 (VIC channel) Using gradient diluted Dry blood spot DNA

The genomic DNA of the dried blood spots of known RPP30 copy number was subjected to gradient dilution according to the method of Table 25.

Table 49: testing linear range of VIC channels in quadruple systems with gradient diluted dry blood spot DNA samples

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Step 4: testing of linear range of SMN1 (CY 5 channel) using gradient diluted dry blood spot DNA

The genomic DNA of the dried blood spots with known SMN1 copy number was subjected to gradient dilution.

Table 50: gradient dilution of known SMN1 copy number of dried blood spot gDNA

Table 51: testing the linear Range of CY5 channels in a quadruple System with gradient diluted Dry blood spot DNA samples

Example 7: research on determination of SCID positive judgment value by quadruple system

Step 1: detection results on clinical samples

96 samples (13 SCID positive samples, 83 negative samples, included) for which mutation had been confirmed by second generation sequencing were tested using a SCID quadruplex test system (fluorescent PCR). And analyzing the difference delta Ct between the TREC Ct value and the internal reference RPP30 Ct value through an ROC curve to determine the positive judgment value of the quadruple detection system on the TREC gene. The results are shown in tables 53, 54, 55 and 56, and the ROC curve is shown in FIG. 1.

The results show that: 1) The area under the ROC curve was 0.966 (table 57), indicating that the diagnostic accuracy of the reagent was very good; 2) The best diagnostic threshold is Δct=12.50, at which time the Sensitivity (Sensitivity) is 0.929, i.e. 92.9%; the specificity was (1-0.012), i.e., 98.8%, with the highest value added (tables 58, 59). Temporarily, a positive determination was made as to delta ct=12.50, and SCID test results are explained in the following table (table 52),

table 52: SCID criterion

Table 53:96 cases of clinical sample detection results-1

Table 54:96 cases of clinical sample detection results-2

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Table 55:96 clinical samples detection result-3

51	Negative of	36.91	37.62	29.69	7.22	7.93
							52	Negative of	36.17	38.25	29.38	6.79	8.87
53	Negative of	31.84	33.12	25.21	6.63	7.91
							54	Negative of	31.7	33.11	25.44	6.26	7.67
55	Negative of	31.33	33.39	25.4	5.93	7.99
							56	Negative of	30.91	32.55	24.7	6.21	7.85
57	Negative of	30.82	31.81	25.07	5.75	6.74
							58	Negative of	30.75	31.23	24.94	5.81	6.29
59	Negative of	32.52	33.42	24.87	7.65	8.55
							60	Negative of	30.93	31.95	25.24	5.69	6.71
61	Negative of	32.04	31.97	25.14	6.90	6.83
							62	Negative of	31.4	32.98	25.09	6.31	7.89
63	Negative of	30.92	30.87	24.98	5.94	5.89
							64	Negative of	31.29	32.24	24.64	6.65	7.60
65	###	31.51	32.52	24.92	6.59	7.60
							66	Negative of	29.41	31.05	24.54	4.87	6.51
67	Negative of	32.73	32.92	26.52	6.29	6.36
							68	Negative of	32.91	31.61	26.59	6.39	4.98
69	Negative of	33.1	33.62	26.95	6.23	6.62
							70	Negative of	32.51	33.14	26.54	6.04	6.56
71	Negative of	33.55	31.55	27.53	6.09	3.98
							72	Negative of	31.82	30.38	25.94	5.96	4.4
73	Negative of	33	33.09	26.6	6.48	6.46
							74	Negative of	30.5	31.9	25.25	5.33	6.61
75	Negative of	31.55	33.77	26.35	5.23	7.34

Table 56:96 cases of clinical sample detection results-4

76	Negative of	31.96	34.92	26.3	5.69	8.54
							77	Negative of	30.41	31.31	25.51	4.93	5.72
78	Negative of	33.18	32.95	26.93	6.29	5.94
							79	Negative of	30.84	33.83	25.95	4.92	7.8
80	Negative of	32.14	32.98	24.53	7.64	8.37
							81	Negative of	30.73	31.83	24.96	5.8	6.78
82	Negative of	30.3	32.08	24.24	6.1	7.76
							83	Negative of	31.74	34.09	26.52	5.25	7.49
84	Negative of	32.53	32.33	26.75	5.81	5.5
							85	Negative of	32.36	33.16	26.73	5.67	6.34
86	Negative of	32.42	32.71	26.95	5.51	5.68
							87	Negative of	32.72	32.32	26.26	6.5	5.98
88	Negative of	31.88	35.67	26.18	5.73	9.41
							89	Negative of	32.37	32.29	26.26	6.15	5.95
90	Negative of	32.02	32.71	26.02	6.04	6.61
							91	Negative of	31.88	31.15	26.89	4.99	4.21
92	Negative of	31.92	32.72	26.38	5.55	6.29
							93	Negative of	31.61	32.05	26.3	5.31	5.7
94	Negative of	30.7	31.79	25.38	5.31	6.36
							95	Negative of	31.81	33.69	25.95	5.86	7.69
96	Negative of	28.98	31.8	24.29	4.69	7.46

Step 2: ROC curve production

Fig. 1 is a ROC graph.

Table 57: area under curve

Table 58: coordinates of curve-1

Table 59: coordinates of curve-2

Step 3: verification of SCID Positive judgment value

According to the proposed positive judgment value judging method, 18 samples with related gene mutation conditions confirmed by secondary sequencing are detected, and the result shows that the detection result of the kit is consistent with the secondary sequencing conclusion, so that the positive judgment method set by the kit is reasonable. The results of the verification are shown in Table 60.

Table 60: positive judgment value verification result

Example 8: research on determination of SMA positive judgment value by quadruple system

Step 1, detection results of clinical samples

Samples 28 (including 7 SMA positive samples and 21 negative samples) for which mutation had been confirmed by second generation sequencing were tested using the SCID quadruple test system (fluorescent PCR method). And analyzing the difference delta Ct between the SMN1 Ct value and the internal reference RPP30 Ct value through a ROC curve to determine the positive judgment value of the quadruple detection system on the SMN1 gene.

The results are shown in Table 62 and the ROC curve is shown in FIG. 2.

The results show that: 1) The area under the ROC curve was 1.0 (table 63), indicating that the diagnostic accuracy of the reagent was very good; 2) The best diagnostic threshold is Δct=8.50, where the Sensitivity (Sensitivity) is 1.0, i.e. 100%; the specificity was 1.0, i.e. 100%, with the highest value added (Table 64). Temporarily, Δct=8.0 is assumed as a positive judgment value, and the detection results are explained in the following table (table 61),

table 61: criterion for SMA

Table 62: test results of 28 clinical samples

Sample ID	SMN1 CT value	RPP30 CT value	Delta Ct value	Detection result	(gold standard) second generation sequencing results
						123	NaN	25.47	-	Positive and negative	SMN1:0 copy
124	NaN	25.95	-	Positive and negative	SMN1:0 copy
						125	NaN	26.88	-	Positive and negative	SMN1:0 copy
126	NaN	25.49	-	Positive and negative	SMN1:0 copy
						127	NaN	25.56	-	Positive and negative	SMN1:0 copy
128	NaN	25.28	-	Positive and negative	SMN1:0 copy
						129	NaN	25.37	-	Positive and negative	SMN1:0 copy
130	26.31	26.09	0.22	Negative of	SMN1:2 copies
						131	26.71	25.66	1.05	Negative of	SMN1:1 copy
132	24.7	24.61	0.09	Negative of	SMN1:2 copies
						133	26.46	26.43	0.03	Negative of	SMN1:2 copies
134	25.07	25.15	-0.08	Negative of	SMN1:2 copies
						135	24.96	25.61	-0.65	Negative of	SMN1:3 copies
136	26.49	26.44	0.05	Negative of	SMN1:2 copies
						137	25.01	24.92	0.09	Negative of	SMN1:2 copies
138	26.05	25.15	0.9	Negative of	SMN1:1 copy
						139	25.06	24.88	0.18	Negative of	SMN1:2 copies
140	26.02	25.87	0.15	Negative of	SMN1:2 copies
						141	24.09	24.19	-0.1	Negative of	SMN1:2 copies
142	23.7	23.64	0.06	Negative of	SMN1:2 copies
						143	26.02	26.1	-0.08	Negative of	SMN1:2 copies
144	22.29	22.34	-0.05	Negative of	SMN1:2 copies
						145	24.94	25.06	-0.12	Negative of	SMN1:2 copies
146	23.84	24.04	-0.2	Negative of	SMN1:2 copies
						147	22.98	22.97	0.01	Negative of	SMN1:2 copies
148	23.86	23.97	-0.11	Negative of	SMN1:2 copies
						149	23.71	23.79	-0.08	Negative of	SMN1:2 copies
150	23.72	23.59	0.13	Negative of	SMN1:2 copies

Step 2: ROC curve production

Fig. 2: ROC curve.

Table 63: area under curve

Table 64: coordinates of a curve

Step 3: verification of SMA Positive judgment value

According to the proposed positive judgment value judging method, 20 samples with related gene mutation conditions confirmed by secondary sequencing are detected, and the result shows that the detection result of the kit is consistent with the secondary sequencing conclusion, so that the positive judgment method set by the kit is reasonable. The verification results are shown in Table 65.

Table 65

Sample numbering	Second generation sequencing results	Detection result of kit	Ct _CY5	Ct _VIC	ΔCt(Ct _CY5- Ct _VIC )
						151	SMN1:0 copy	Positive and negative	NaN	33.85	-
152	SMN1:0 copy	Positive and negative	NaN	34.15	-
						153	SMN1:0 copy	Positive and negative	NaN	34.10	-
154	SMN1:0 copy	Positive and negative	NaN	34.20	-
						155	SMN1:0 copy	Positive and negative	NaN	33.90	-
156	SMN1:1 copy	Negative of	34.36	32.86	1.5
						157	SMN1:1 copy	Negative of	34.07	33.28	0.79
158	SMN1:1 copy	Negative of	33.86	32.22	1.64
						159	SMN1:1 copy	Negative of	34.21	32.69	1.52
160	SMN1:1 copy	Negative of	33.52	32.25	1.27
						161	SMN1:2 copies	Negative of	34.78	34.62	0.16
162	SMN1:2 copies	Negative of	34.28	33.90	0.38
						163	SMN1:2 copies	Negative of	34.74	33.83	0.91
164	SMN1:2 copies	Negative of	33.97	34.09	-0.12
						165	SMN1:2 copies	Negative of	34.43	33.92	0.51
166	SMN1:3 copies	Negative of	33.96	34.61	-0.65
						167	SMN1:3 copies	Negative of	33.52	34.34	-0.82
168	SMN1:3 copies	Negative of	33.38	34.14	-0.76
						169	SMN1:3 copies	Negative of	33.10	33.95	-0.85
170	SMN1:3 copies	Negative of	32.95	33.85	-0.9

Example 9: research on copy number detection limit of each fluorescence channel of quadruple fluorescence quantitative system

According to the most preferred S1-S5 operation steps of the invention, detection of a detection limit research experiment is carried out on a quadruple detection system.

S1: and extracting nucleic acid from the dried blood spots. The step S1 specifically comprises the following steps: extracting nucleic acid by using a dry blood spot nucleic acid extraction kit (magnetic bead method, DP 344) of radix et rhizoma Nardostachyos, taking 2-3 pieces of dry blood spot samples with the diameter of 3mm, treating with 400uL of lysate and 15uL of magnetic beads at 75 ℃ for 45min, transferring the supernatant into a new centrifuge tube, adding 400uL of nucleic acid binding solution and 10uL of magnetic beads, mixing at normal temperature for 10min, enriching the magnetic beads with a magnetic rack, washing the magnetic beads twice with washing solution 1, washing the collected magnetic beads twice with washing solution 2, airing the magnetic beads, adding 50uL of eluent, incubating at 75 ℃ for 10min, enriching and discarding the magnetic beads with the magnetic rack, sucking the supernatant into the centrifuge tube to 1.5mL, namely the obtained DNA, and freezing and preserving at-20 ℃.

S2: and (5) preparing a reaction solution. The step S2 specifically comprises the following steps: and (3) subpackaging the PCR reaction liquid according to the number n of samples to be tested (negative control and positive control are needed), and subpackaging the PCR reaction liquid into PCR reaction tubes/plates according to 15 mu l/tube. More preferably, the enzyme premix used in the present kit is 2x Taqman Fast Advanced Master Mix (thermosusher, cat# 4444557). More preferably, the primer probe used in the kit is a sequence synthesized autonomously by Shanghai biological organisms.

The preparation of the PCR reaction liquid in the step S2 is shown in Table 66, and the sequences of the primers and probes used for preparing the PCR reaction liquid are shown in Table 67.

Table 66: preparation of PCR reaction solution

Raw materials	24 people's kit
		2×Taqman Fast Advanced Master Mix	250μl
TREC-F(100μM)	0.875μl
		TREC-R(100μM)	0.875μl
TREC-P(100μM)	0.425μl
		KREC-F(100μM)	1.5μl
KREC-R(100μM)	1.5μl
		KREC-P(100μM)	1.25μl
RPP30-F(100μM)	0.375μl
		RPP30-F(100μM)	0.375μl
RPP30-F(100μM)	0.3μl
		SMN1-F(100μM)	0.75μl
SMN1-R(100μM)	0.75μl
		SMN1-P(100μM)	0.375μl
H ₂ O	115.65μl

Table 67: primer probe nucleic acid standard sequence table

S3: and (5) sample adding. The step S3 specifically comprises the following steps: and respectively adding the nucleic acid extracted from the sample to be detected, the nucleic acid extracted from the negative quality control product and the positive quality control product into the PCR reaction liquid, covering the tube cover or sealing the film, and carrying out instantaneous centrifugation for a plurality of seconds to concentrate the liquid to the bottom of the tube and transfer the liquid into a nucleic acid amplification region. More preferably, the total system of the single reaction is formulated as in table 68:

table 68

PCR reaction solution dosage	Template dosage	Total system
			15μl	5μl	20μl

S4: qPCR amplification. The step S4 specifically comprises the following steps: (1) placing a PCR reaction tube/plate into a sample groove of a quantitative PCR instrument, setting positive control, negative control and unknown samples according to corresponding names, and setting sample names and detection target names; (2) fluorescence detection channel selection: FAM channel detection TREC was selected, and the quenching group was "none"; selecting HEX/VIC channel to detect internal reference; the quenching group selects "MGB"; selecting NED channels to detect KREC; the quenching group selected "none" reference dye Passive Reference selected "ROX"; selecting a CY5 channel to detect SMN1; the quenching group selected "none" reference dye Passive Reference selected "ROX" (3) reaction procedure set-up; (4) the reaction system was selected to be 20. Mu.L; (5) and after the setting is finished, storing a file and running a reaction program. The reaction procedure used in step S4 is as shown in table 69:

Table 69

The positive quality control is plasmid PUC57-RPP30 containing an amplified fragment of the reference gene of RPP30.

The negative quality control is plasmid PUC57-TREC-KREC-RPP30-SMN1 containing TREC, KREC, SMN and RPP30 reference gene amplified fragment.

S5: and (5) data analysis. The step S5 specifically comprises the following steps: (1) results analysis condition settings, adjusting ABI7500 baseline (automatic setting of selection), threshold line manually set the same threshold for FAM, NED, VIC, CY5 four fluorescence channels: 0.1-0.12; (2) judging a quality control product; (3) and judging the detection result.

The quality control criteria used in step S5 are as shown in table 70.

Table 70: quality control standard

The specific scheme is as follows:

plasmid samples (PUC 57-TREC-KREC-RPP30-SMN 1) were quantified by standard curve, plasmid DNA was diluted with 1 XTE buffer to 170 copies/. Mu.L, 85 copies/. Mu.L, 43 copies/. Mu.L, 21 copies/. Mu.L, 11 copies/. Mu.L, 5 copies/. Mu.L, 2.5 copies/. Mu.L and 1 copies/. Mu.L, 5. Mu.L of plasmid templates of different copy numbers were taken, and detected using a quality-check-qualified triple system detection kit (fluorescent PCR method), and the range of the lowest detection limit (. Gtoreq.95% detection rate) was estimated preliminarily (Table 72). And (5) taking the plasmid template with low copy number for 20 times of repeated detection, and calculating the detection rate of 20 times of detection results. When the detection rate of the detection results of the concentrations for 20 times is more than or equal to 95 percent (the fluorescence channel to be detected has detection signals, the Ct value is less than 40.0), and the Ct value CV is less than or equal to 5 percent, the lowest concentration meeting the condition is taken as the detection limit, and if the concentrations meet the condition, the concentration continues to be diluted downwards and the lowest concentration meeting the condition is detected as the detection limit. In performing this evaluation, a panel of operators used the same template and the same lot number reagents to perform the detection on an ABI7500 fluorescent quantitative PCR instrument.

The result shows that the detection limit of the kit on the 13 copies/reaction plasmid template is 95%, and the Ct value CV is less than or equal to 5%. The estimated limit of detection for the PUC57-TREC-KREC-RPP30-SMN1 plasmid template was 13 copies per reaction tube, and the detection results are shown in Table 71.

The detection limit study was further performed on TREC (FAM), KREC (NED), SMN1 (CY 5) in a quadruple detection system using the dried blood spot DNA sample as a template. The TREC, KREC, SMN copy number in SCID & SMA negative dry blood spot DNA samples was determined by using a fixed plasmid standard and standard curve (Table 73-Table 74, table 75, table 76, table 77 and Table 78). And diluting the DNA sample of the fixed dry blood spot to the vicinity of the detection limit by using enzyme-free water, and calculating the detection rate of 20 detection results. When the detection rate of the detection result of each concentration for 20 times is more than or equal to 95 percent (the detection signals of the fluorescence channel to be detected are detected, TREC and KREC Ct values are less than 40.0, SMN1 Ct value is less than or equal to 35.0, and Ct value CV is less than or equal to 5 percent), the lowest concentration meeting the condition is taken as the detection limit. If both conditions are met, continuing to dilute downwards and detecting the lowest concentration meeting the conditions as a detection limit. In performing this evaluation, a panel of operators used the same template and the same lot number reagents to perform the detection on an ABI 7500 fluorescent quantitative PCR instrument.

The results show that the detection limits of the dried blood spot DNA samples TREC and KREC are 8 copies and 9 copies/reaction respectively; the detection limit of SMN1 was 51 copies/reaction (0.31 ng gDNA/reaction), and the detection results are shown in tables 75 to 78, 81 and 82.

Step 1: minimum detection limit study of tandem plasmid templates in quadruple system

Table 71: ct values detected in quadruple system for tandem plasmid DNA of different copy numbers

Table 72: lowest limit of detection experiment result of quadruple system on plasmid template

Step 2: research on TREC and KREC detection limit in dry blood spot DNA sample by quadruple system

The TREC and KREC copy numbers in negative dry blood spot DNA samples were quantified by using a fixed plasmid standard and standard curve. The DNA sample of the fixed dry blood spot was diluted to the vicinity of the detection limit by using enzyme-free water, and the dilution method is shown in the table. Samples with TREC and KREC copy numbers close to the detection limit are used as templates.

Table 73: dry blood spot DNA 1 sample concentration, copy number and gradient dilution method

Table 74: ct value detected in quadruple system of gradient diluted dry blood spot DNA sample

Table 75: dry blood spot DNA sample TREC and KREC minimum detection limit detection result (concentration 3)

Table 76: dry blood spot DNA sample TREC and KREC minimum detection limit detection result (concentration 4)

Table 77: dry blood spot DNA sample TREC and KREC minimum detection limit detection result (concentration 5)

Table 78: dry blood spot DNA sample TREC and KREC minimum detection limit detection result (concentration 6)

Step 3: research on detection limit of SMA (shape memory alloy) of dry blood spot DNA sample by quadruple system

RPP30, SMN1 copy number of DNA product was quantified by using a fixed plasmid standard and standard curve to quantify dry blood spot samples containing single copy SMN 1. The dried blood spot DNA sample was diluted 2-fold with enzyme-free water to the limit of detection, and the dilution method is shown in the table. The DNA sample with gradient dilution is used as a template to detect in a quadruple system, and the DNA sample with SMN1 copy number close to the detection limit is used as the template to study the detection limit.

Setting of SMN1 detection limit: repeating detection of 20 tubes on ABI7500 for the same DNA template, manually setting CY5 (SMN 1) and VIC (RPP 30) fluorescence channel threshold to 0.1-0.12, and equalizing the two, wherein the detection limit is that for DNA samples with higher than specific concentration (copy number), the sample Ct is more than or equal to 95% (19/20) _SMN1 Less than or equal to 35.0 and Ct _SMN1 -Ct _RPP30 CV with Ct value less than or equal to 8.0 and less than or equal to 5%.

Results: the detection limit of SMA in the quadruple system was 0.31 ng/. Mu.L (51 copies/reaction).

Table 79: dry blood spot DNA 2 sample concentration, copy number and gradient dilution method

Table 80: ct value detected in quadruple system of gradient diluted dry blood spot DNA sample

Table 81: detection result of minimum detection limit of SMN1 and RPP30 of dry blood spot DNA sample

Table 82: detection result of minimum detection limit of SMN1 and RPP30 of dry blood spot DNA sample

Example 9: investigation of analytical precision

The specific scheme is as follows: the same SCID negative samples (medium and low concentrations) and SCID positive samples (medium concentration) were each tested on the same instrument (A) daily by the same 1 operator (P1) using the same lot number of reagents, and the test was continued for 10 days, with 10 replicates.

Acceptable standards: the results of the negative samples repeated for 10 days should be negative; calculating the detection precision from 10 Ct values of the same sample detected each time, and meeting the precision requirement when the variation coefficient CV of the Ct values is less than 5%; the results of 10 days repeated positive samples should be positive; and calculating the precision in the batch from the Ct values of 6 internal reference genes of the same sample detected each time, and meeting the precision requirement in the batch when the variation coefficient CV of the Ct values is less than 5%.

The results show that the negative coincidence rate of the SCID and SMA negative samples with medium and low concentrations is 100 percent (table 83, table 84, table 85, table 86 and table 87) after 10 days of continuous detection, and the variation coefficient CV of Ct value is less than or equal to 5 percent; for SCID positive samples with medium concentration and SMA positive samples with medium and low concentrations, the positive coincidence rate is 100% after 10 days of continuous detection, and the variation coefficient CV of the internal reference RPP30 and KREC Ct values is less than or equal to 5% (TREC of most samples has no amplified signal) (tables 88, 89 and 90). The quadruple detection reagent has good precision.

Table 83: analysis precision-SCID/SMA negative compliance statistics (first day to second day)

Table 84: analysis precision-SCID/SMA negative compliance statistics (third day to fourth day)

Table 85: analysis precision-SCID/SMA negative compliance statistics (fifth day to sixth day)

Table 86: analysis precision-SCID/SMA negative compliance statistics (seventh day to eighth day)

Table 87: analysis precision-SCID/SMA negative compliance statistics (day nine to day ten)

Table 88: analysis precision-SCID positive compliance statistics

Table 89: analysis precision-SMA positive coincidence rate statistical result-1

Table 90: analysis precision-SMA Positive coincidence Rate statistical result-2

In general, the examples herein determine the sequence of TRECs, KRECs, SMN gene and reference gene test primers and probes. According to the structural characteristics of TREC and KREC recombination rings, primers are designed at the upstream and downstream of recombination sites, and probes are designed near the recombination sites, so that the specificity of TREC and KREC ring detection is ensured; the upstream and downstream primers of the SMN1 respectively comprise SNP loci of SMN1 genes at exon 7 and intron 7, and LNA modification is introduced into the primers to improve the Tm value and the specificity of recognition of a template; the highly conserved region of the single copy gene RPP30 was selected to design the primer and probe sequences of the reference gene. The copy numbers of TREC, KREC and SMN1 genes in the number of blood cells per unit can be calculated by comparing the amplified Ct values with those of the reference genes (delta Ct method). The amplification specificity of the designed primers and probes is verified by testing the amplification effect of a single system and a multiple system on blank, specific and non-specific templates, and a quadruple detection system is constructed by selecting the combination with optimal specificity and amplification effect on the basis.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings, and equivalents thereof fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. The detection kit is characterized by comprising a primer pair 1 and a probe 1 for detecting TRECs, a primer pair 2 and a probe 2 for detecting KRECs, and a primer pair 3 and a probe 3 for detecting and SMN1 genes, or further comprising an internal reference primer pair and an internal reference probe for detecting internal reference genes;

wherein,,

2. The test kit according to claim 1, characterized in that it has one or more of the following technical features:

3. The detection kit according to claim 2, wherein the 15 th and 23 rd nucleotides of the upstream primer 3 have LNA modification, and the 13 th nucleotide of the downstream primer 3 has LNA modification.

4. The test kit according to claim 1, characterized in that it has one or more of the following technical features:

(1) The probe 1 is shown as SEQ ID No. 4;

(3) The probe 3 is shown as SEQ ID No. 24.

5. The detection kit according to claim 4, wherein the 12 th and 14 th nucleotides of the probe 3 have LNA modifications.

6. The test kit according to any one of claims 1 to 5, wherein the reference gene is the human RPP30 gene.

7. The detection kit according to any one of claims 1 to 5, wherein the detection probe and the reference probe are each independently labeled with a fluorescent reporter group and a fluorescent quenching group, and the fluorescent reporter groups labeled are different between different detection probes and between a detection probe and a reference probe.

8. A method for detecting TRECs, KRECs and SMN1 genes for non-diagnostic purposes, characterized in that the method comprises the steps of:

detecting a nucleic acid sample to be detected by using the detection kit according to any one of claims 1 to 7, and judging whether the copy numbers of TRECs, KRECs and SMN1 genes in the nucleic acid sample to be detected are abnormal according to the detection result.

9. The method for detecting TRECs, KRECs and SMN1 genes for non-diagnostic purposes according to claim 8, wherein the annealing temperature for amplification is 58℃to 62 ℃.

10. The method for detecting TRECs, KRECs and SMN1 genes for non-diagnostic purposes according to claim 9 or 10, wherein determining whether the copy numbers of TRECs, KRECs and SMN1 genes in the nucleic acid sample to be detected are abnormal based on the detection result comprises:

the Ct values corresponding to the probe 1, the probe 2 and the reference probe are respectively referred to as Ct _{Detection 1} 、Ct _{Detection 2} And Ct _{Internal reference} Calculation of

ΔCt ₁ ＝|Ct _{Detection 1} -Ct _{Internal reference} |，ΔCt ₂ ＝|Ct _{Detection 2} -Ct _{Internal reference} |，

(1)Ct _{internal reference} ≤27，ΔCt ₁ ≤10.0，ΔCt ₂ ≤10.0；

(2)Ct _{Internal reference} ≤27，ΔCt ₁ >12.5; alternatively, ct _{Internal reference} Ct shows "undetermined". Ltoreq.27; alternatively, ct _{Internal reference} Less than or equal to 27, although Ct values are shown, no obvious amplification curve is generated;

the method comprises the steps of,

(5)Ct _{Internal reference} ≤300，ΔCt ₃ ≤8.0；