CN109709248B - Application of reagent and adsorption column in preparation of kit for detecting to-be-detected object in dried blood slice sample - Google Patents
Application of reagent and adsorption column in preparation of kit for detecting to-be-detected object in dried blood slice sample Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting an object to be detected in a dried blood slice sample and application thereof. The method for detecting the substance to be detected in the dried blood slice sample comprises the steps of separating a metabolite from the dried blood slice sample; and a step of separating genetic material from the dried blood sample. The method of the invention allows the detection of a plurality of different analytes to be detected simultaneously or with a smaller amount of sample. The invention can shorten the time for screening and diagnosing the neonatal diseases and can use a smaller amount of samples.
Description
Technical Field
The present invention relates to the analysis of dried blood slice samples, in particular to a method and a kit for detecting an analyte to be detected, in particular a metabolite and a genetic material simultaneously in the dried blood slice samples, and uses thereof.
Background
The newborn disease screening refers to the group screening of certain seriously-harmful congenital genetic metabolic diseases in the newborn period, so that children patients can be diagnosed and treated early, the occurrence of intelligent disability is avoided, and the population quality is improved. The disease often lacks specific symptoms in the neonatal period, and once symptoms appear, the infant suffers irreversible damage to the central nervous system, and loses the good treatment opportunity. The indexes of biochemistry, metabolism and the like of the infant in the blood of the newborn stage are changed, so that the early diagnosis can be made by utilizing a laboratory detection method.
Currently, dry biological fluid samples, such as Dry Blood Spots (DBS), are becoming increasingly popular in clinical trials for neonatal disease screening. The collection site for the clinical trial may be on-site sampling of blood spots (or other types of biological fluid spots) which, after drying of the blood sheet, may be transported at a lower cost than liquid samples. In addition, since it is capable of detecting a plurality of amino acids and carnitine and the like at the same time, tandem mass spectrometry has been widely used in recent years, and many developed countries use this technique to screen newborn inherited metabolic diseases, such as phenylketonuria, tromethamine, maple syrup urine disease, lactic acidosis, urea cycle disorder and the like. The principle of tandem mass spectrometry (MS/MS) is that two mass spectrometers are connected in series via a collision cell in order to improve the specificity and sensitivity of the detection. MS/MS can detect dozens of amino acids at the same time, can assist in diagnosing dozens of genetic metabolic diseases related to the amino acids, and the detected disease spectrum is remarkably expanded. The accuracy of diagnosis of certain diseases can be improved by calculating the ratio of amino acid or carnitine, so that one of the characteristics superior to other technologies is that the false negative and false positive of screening diagnosis can be greatly reduced, especially for diseases which are already screened conventionally.
After screening for diseases in newborn infants, the infant patients with high potential or risk need to be further diagnosed, and the current method for diagnosis in the early stage is to detect the same or different batches of samples again by using the same detection means (e.g., mass spectrometry). For example, when the content or ratio of an amino acid or carnitine is abnormal and there is a disease risk, the same method is required to be performed again on the same batch of dry blood sample to eliminate errors caused by the operation, if the content or ratio of the amino acid or carnitine is still abnormal, the sample of the subject is required to be collected again, the mass spectrum is used again to perform the same detection for the second time to eliminate errors caused by the sample abnormality. Although the error caused by operation and sample abnormality can be greatly reduced by means of detection, the error caused by the detection means can not be eliminated. In addition, this method of confirming diagnosis requires the re-collection of a sample of the same subject. Since the subjects are usually scattered and need to be collected on site, or the potential children are needed to arrive at the designated sample collection site, it is very inconvenient for remote areas or areas with inconvenient transportation conditions, and it is also very inconvenient to call the newborn baby to the designated site. Thus, a long time is usually required from initial screening to confirmation, which is very disadvantageous for early confirmation and timely intervention.
In general, the amount of blood contained in a dried blood sample is low and is not sufficient for detection by different multiplex detection means. While there are currently protocols that employ different multivariate techniques for determining diagnosis, these protocols employ different batches of samples. Due to differences between different batches of samples, for example, samples from different time periods of the subject may vary. In addition, even the same sample may cause different variations in the components under different storage conditions, leading to differences in the diagnosis results. Thus, existing diagnostic protocols do not meet the requirements of increasing precision.
Disclosure of Invention
In order to solve at least part of the above technical problems, the present invention provides a solution for performing the detection of a plurality of different analytes simultaneously or using a smaller amount of sample. The time for screening and diagnosing the neonatal diseases can be shortened by the scheme of the invention, and a smaller amount of samples can be used. Specifically, the present invention includes the following.
In a first aspect of the invention, there is provided a non-diagnostic method for detecting an analyte in a dried blood sample, comprising the steps of:
(1) a step of separating metabolites from the dried blood sample; and
(2) a step of separating genetic material from the dried blood sample.
In certain embodiments, step (1) comprises treating the dried blood sample with a solution comprising methanol, an amino acid, and an isotopic carnitine internal standard and succinylacetone, and separating to obtain a first supernatant and a precipitate, and step (2) comprises treating the precipitate with a solvent comprising tris and a cation remover, followed by addition of a phenol chloroform mixture for further treatment, and centrifuging to obtain a second supernatant.
In certain embodiments, the non-diagnostic method for detecting an analyte in a dried blood sample further comprises (3) the step of purifying the genetic material from the second supernatant using an adsorption column to obtain purified genetic material.
In certain embodiments, the non-diagnostic method for detecting an analyte in a dried blood sample further comprises (4) the step of analyzing the first supernatant using tandem mass spectrometry.
In certain embodiments, wherein the subject of tandem mass spectrometry comprises alanine, arginine, citrulline, glycine, methionine, leucine + isoleucine + hydroxyproline, ornithine, phenylalanine, proline, tyrosine, valine, succinylacetone, free carnitine, acetyl-carnitine, propionyl-carnitine, malonyl-carnitine + 3-hydroxybutyryl-carnitine, butyryl-carnitine, methylmalonyl-carnitine + 3-hydroxyisovaleryl-carnitine, isovaleryl-carnitine, methylcrotonyl-carnitine, glutaryl-carnitine + 3-hydroxyhexanoyl-carnitine, caproyl-carnitine, adipoyl-carnitine, octanoyl-carnitine, octenoyl-carnitine, decanoyl-carnitine, decenoyl-carnitine, dodecanoyl-carnitine, dodecenoyl-carnitine, tetradecanoyl-carnitine, tetradecenoyl-carnitine, Tetradecadienoylcarnitine, 3-hydroxytetradecanoyl carnitine, hexadecanoyl carnitine, hexadecenoyl carnitine, 3-hydroxyhexadecanoyl carnitine, octadecanoyl carnitine, octadecadienoyl carnitine, 3-hydroxyoctadecanoyl carnitine and 3-hydroxyoctadecanoyl carnitine.
In certain embodiments, the non-diagnostic method for detecting an analyte in a dried blood sample further comprises (5) the step of constructing a secondary sequencing library using the purified genetic material.
In a second aspect of the invention, a kit for detecting an analyte in a dried blood sample is provided, comprising a first reagent composition for treating a metabolite in the dried blood sample and a second reagent composition for treating genetic material in the dried blood sample;
wherein the first reagent composition comprises separately stored solutions containing an amino acid and an acylcarnitine isotope internal standard, a methanol-containing solvent, and a acetone succinate-containing solution; the second reagent composition includes a solvent containing tris and a cation remover, a phenol chloroform mixture, and a genetic material amplification mixture, which are stored separately.
In a third aspect of the invention, there is provided the use of a combination of a first reagent composition and a second reagent composition in the manufacture of a kit for neonatal disease or condition screening, wherein the first reagent composition comprises separately stored a solution comprising an amino acid and an acyl carnitine isotope internal standard, a methanol-containing solvent and a acetone succinate-containing solution; the second reagent composition includes a solvent containing tris and a cation remover, a phenol chloroform mixture, and a genetic material amplification mixture, which are stored separately.
The method of the invention carries out the detection of a plurality of detection means on the same sample, thereby obtaining the diagnosis result more timely and rapidly. In addition, the method can simultaneously or use a smaller amount of samples to detect a plurality of different objects to be detected, thereby realizing the aim of performing multi-element detection on the same sample. Preferably, the time for screening and diagnosis of neonatal diseases can be shortened by the present invention, and a smaller amount of sample can be used.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
The dry blood slice sample refers to a sample obtained by dripping a whole blood sample collected at a sample collection site into a dry blood slice. Preferably, the dried blood sheet sample is a spot formed by blood on an absorbent substrate (e.g., filter paper), i.e., a DBS sample. The dried blood sample of the present invention may be prepared by any known technique. An exemplary preparation method is as follows: a drop of blood (typically venous or peripheral blood) is applied to an absorbent substrate of suitable composition. The blood is applied in one drop, is not repeatedly dropped, and is dried in the vent for a period of time (e.g., hours) sufficient to form an array of circular dried blood spot spots on the substrate. The substrate containing the spots can then be stored in a plastic container and transported as needed without the need for refrigeration. In testing, the dried blood sheet blood spots can be separated from the bulk substrate by punching the dried blood sheet blood spots to create individual discs of dried blood sheet blood spots.
The "substance to be detected" of the present invention refers to any substance that can be used as an index for screening neonatal disease, i.e., a marker. The analyte of the present invention includes a variety of different types of substances. Preferably, at least metabolites and genetic material of the body are included. The genetic material of the present invention preferably refers to genetic material derived from body fluids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The present invention may use one or a combination of both of DNA or RNA.
In a first aspect of the invention, a method, sometimes referred to simply as the "method of the invention", is provided for detecting an analyte in a dried blood sample. The method of the present invention is a method for detecting at least two different types of analytes, and in order to detect the two different types of analytes, the prior art requires different treatments for different samples or parts of samples. For example, it may be desirable to take multiple samples of the same subject, or to isolate the same sample for multiple different treatments.
The method for detecting an analyte in a dried blood sample of the present invention comprises the steps of (1) separating a metabolite from the dried blood sample and (2) separating a genetic material from the dried blood sample. Optionally, further comprising (3) a step of purifying the genetic material from the second supernatant using an adsorption column; (4) a step of analyzing the metabolites by tandem mass spectrometry; (5) and (3) constructing a second-generation sequencing library by using genetic materials. The respective steps are explained in detail below.
The step (1) is a step of separating metabolites from the dried blood sample. Preferably, the separating step comprises treating the dried blood sample with a solution containing methanol, an isotopic internal standard of amino acids and acylcarnitines, acetone succinate, and separating to obtain a first supernatant and a precipitate. Unlike prior methods, the first supernatant contains metabolites and the precipitate can be further used for the isolation of genetic material. In order to better reflect the accurate content and level of metabolites, especially arginine and carnitine, in the blood sample, it is preferable to use a centrally perforated dried blood sample, since the centrally perforated dried blood sample is more suitable for the separation of step (1) of the present invention. In certain embodiments, the treatment conditions in step (1) comprise shaking at 40-50 ℃ for 30-60 minutes. The above-mentioned treatment conditions are essential for the isolation of the metabolites. If the temperature is too low, the metabolite is not sufficiently dissolved, and even an effective amount of the metabolite is obtained. On the other hand, if the temperature is too high, structural changes, even decomposition, of the metabolite may be caused. In addition, the treatment in step (1) needs to be performed under a closed condition to prevent the volatilization of methanol and the influence of dissolution. Obtaining a first supernatant containing metabolites and a precipitate containing genetic materials after the treatment of the step (1).
The step (2) is a step of separating genetic material from the dried blood sample. Preferably, the dried blood sample in step (1) is the same sample as the dried blood sample in step (2), thereby reducing the amount of sample used. More preferably, the step (2) comprises treating the precipitate obtained in the step (1) with a solvent containing tris and a cation remover, then adding a phenol-chloroform mixture to the precipitate-dissolved solution for further treatment, obtaining a second supernatant after centrifugation, and separating the genetic material from the second supernatant. The solvent containing tris and the cation remover in step (2) is an aqueous solution having a pH of 7.5 to 9, preferably 8.0. An aqueous solution of tris can be adjusted to a desired pH range using hydrochloric acid and then the desired amount of cation remover is added. Examples of the cation remover include metal salts of ethylenediaminetetraacetic acid, such as sodium salts. And (2) adding a phenol-chloroform mixed solution into the precipitate dissolving solution for further treatment. The volume ratio of phenol to chloroform in the phenol-chloroform mixture is preferably 1: 1. Preferably, the phenol/chloroform mixture further contains proteinase K.
The step (3) is a step of purifying the genetic material from the second supernatant using an adsorption column. The adsorption column is preferably CG2 adsorption column. Step (3) may use known reagents and/or known procedures.
The step (4) is a step of analyzing the sample liquid by using tandem mass spectrometry. Step (4) can be carried out by known means. The tandem mass spectrometry object in the step (4) comprises amino acid substances, ketone substances and carnitine substances. Examples of amino acids include alanine, arginine, citrulline, glycine, methionine, leucine + isoleucine + hydroxyproline, ornithine, phenylalanine, proline, tyrosine, valine. Examples of ketones include succinylacetone and the like. Examples of carnitine-based substances include free carnitine, acetyl carnitine, propionyl carnitine, malonyl carnitine + 3-hydroxybutyryl carnitine, butyryl carnitine, methylmalonyl carnitine + 3-hydroxyisovaleryl carnitine, isovaleryl carnitine, methylcrotonyl carnitine, glutaryl carnitine + 3-hydroxyhexanoyl carnitine, hexanoyl carnitine, adipyl carnitine, octanoyl carnitine, octenoyl carnitine, decanoyl carnitine, decenoyl carnitine, decadienyl carnitine, dodecanoyl carnitine, dodecenoyl carnitine, tetradecanoyl carnitine, tetradecenoyl carnitine, 3-hydroxytetradecanoyl carnitine, hexadecanoyl carnitine, 3-hydroxyhexadecanoyl carnitine, octadecanoyl carnitine, Octadecenoylcarnitine, octadecadienylcarnitine, 3-hydroxyoctadecanoylcarnitine, 3-hydroxyoctadecenoylcarnitine, and the like. Preferably, the metabolites of the present invention include both amino acids, ketones and carnitine. For example, the results of all the above metabolites are obtained simultaneously after one test.
The step (5) is a step of constructing a second generation sequencing library by using genetic materials. That is, a method of determining the sequence of DNA by capturing a marker of a newly synthesized end. The construction of the library can be carried out using known techniques. For example, it is carried out using a known kit. In addition, library construction and sequencing can be performed using existing technology platforms. For example, Roche (Roche)/454 FLX, Illuminate/Solexa Genome Analyzer, and Applied Biosystems SOLID system (ABI).
The sequence of steps (1) to (5) in the present invention is not particularly limited, and may be performed in the sequence of steps (1) to (5), may be appropriately adjusted without affecting the object of the present invention, may be performed simultaneously in a plurality of steps, and may include other steps or sub-steps between the steps. It should also be noted that the method for detecting an analyte in a dried blood sample of the present invention can be used for clinical diagnosis, and also can be used for non-diagnostic purposes, such as scientific research and the like.
In a second aspect of the invention, kits are provided for detecting an analyte in a dried blood sample, sometimes referred to herein simply as "kits of the invention". The kit of the invention comprises a first reagent composition for processing metabolites in a dried blood sample and a second reagent composition for processing genetic material in a dried blood sample.
The first reagent composition of the present invention comprises a solution containing an internal isotope of amino acid and acyl carnitine, a methanol-containing solvent, and a acetone succinate, which are separately stored. The kind of amino acid and acylcarnitine isotopes may vary according to need. The solution containing the internal isotope standards of amino acid and acyl carnitine is preferably a methanol solution. These isotopic labels are available commercially. The methanol-containing solvent is preferably methanol and does not contain any isotopic label. The concentration of the solution containing succinylacetone was around 110. mu. mol/L. The separate storage means that the above-mentioned respective solutions or solvents exist separately in a state of being separated from each other. For example, a solution containing an internal standard for an amino acid isotope is stored in the first space and a solution containing an internal standard for an acyl carnitine isotope is stored in the second space, a methanol-containing solvent is stored in the third space, and a solution containing succinylacetone is stored in the fourth space. The first space, the second space, the third space, and the fourth space are different containers (e.g., vials), and are hermetically sealed from light and low temperature. In certain embodiments, the first reagent composition comprises a first vial containing a solution comprising an isotope of an amino acid and a second vial containing a solution comprising an isotope of an acyl carnitine, a third vial containing a methanol solvent, and a fourth vial containing a solution comprising succinylacetone. Preferably, the first reagent composition may further comprise a microplate, an adhesive sealing film and a foil paper.
The second reagent composition of the present invention comprises a solvent containing tris and a cation remover, a phenol chloroform mixture, and a genetic material amplification mixture, which are stored separately. The solvent containing tris and the cation remover is preferably an aqueous solution, more preferably an aqueous solution having a pH of 7.5 to 9, preferably 8.0. Examples of the cation remover include metal salts of ethylenediaminetetraacetic acid, such as sodium salts. The phenol/chloroform mixture is a mixture of phenol and chloroform at a predetermined volume ratio (for example, 1:1), and the phenol may be phenol or the like. The genetic material amplification mixture preferably includes reagents required to construct a second generation sequencing library. For example, primer sequences, marker sequences, capture primers and DNA polymerase required for PCR, dNTPs of various types and ions such as Mg2+And the like. These agents or ingredients are known to those skilled in the art and can be exemplifiedPublications such as molecular cloning, a guide for experiments, fourth edition, cold spring harbor are readily known.
Preferably, the kit of the present invention further comprises instructions for use, wherein instructions, directions or teaching for carrying out the method of the present invention or for using the composition or kit of the present invention are given or taught in the instructions for use.
In a third aspect of the invention, there is provided the use of a combination of a first reagent composition and a second reagent composition in the manufacture of a kit for neonatal disease or condition screening. The first reagent composition and the second reagent composition have been described in detail above and will not be described herein again.
Examples of neonatal diseases or conditions described herein include, but are not limited to, conditions of the amino acid metabolism disorder class, conditions of the organic acidemia class, and conditions of the fatty acid oxidation deficiency class. Examples of conditions in which the amino acid metabolism disorder is a disorder include phenylketonuria, maple syrup urine disease, homocystinuria, citrullinemia type I, citrullinemia type II, argininosuccinase deficiency, tyrosinemia type I, tyrosinemia type II, tyrosinemia type III, homophenylalaninemia, biopterin biosynthetic enzyme deficiency, biopterin regeneration disorder, argininemia, hyperproteinemia, homoornithine blood-hyperblood ammonia-homocitrullinuria syndrome, hyperprolinemia, nonketotic hyperglycinemia, ornithine carbamoyltransferase deficiency, carbamoylphosphate synthase deficiency. Examples of conditions of the category of organic acidemia include isovaleric acidemia, glutaremia type I, 3-hydroxy-3-methylglutaric acidemia, multiple coa carboxylase deficiency, methylmalonic acidemia (methylmalonyl-coa mutase), methylmalonic acidemia (adenosylcobalamin synthase), methylmalonic acidemia combined with homocysteinemia, 3-methylcrotonyl-coa carboxylase deficiency, propionic acidemia, beta ketothiolase deficiency, malonic acidemia, isobutyrylglycinuria, 2-methyl-3-hydroxybutyric aciduria, 2-methylbutyrylglycinuria, 3-methylpentenedionic aciduria, ethylmalonic acid encephalopathy. Examples of fatty acid oxidation deficiency-type conditions include carnitine uptake disorder, carnitine-acyl carnitine translocase deficiency, glutaremia type II, short-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-CoA dehydrogenase deficiency, very-long-chain acyl-CoA dehydrogenase deficiency, short-chain left-3-hydroxyacyl-CoA dehydrogenase deficiency, long-chain left-3-hydroxyacyl-CoA dehydrogenase deficiency, medium-chain acyl-CoA thiolase deficiency, carnitine palmitoyl transferase deficiency type I, carnitine palmitoyl transferase deficiency type II, 2, 4-dienoyl-CoA reductase deficiency, and trifunctional protein deficiency.
Examples
Firstly, mass spectrum detection:
1. punching a specimen: arranging a specimen (a hyperphenylalaninemia sample), punching a hole in the center of a dry blood spot by using a puncher with the diameter of 3.2mm, putting a U-shaped bottom clean micropore plate, and putting only one blood spot in each hole, wherein the front 6 hole holes in each micropore plate are recommended to be respectively set to be 2 blanks, 2 low quality controls and 2 high quality controls;
2. preparing a mixed working solution (hereinafter referred to as working solution) containing methanol, amino acid, acyl carnitine isotope internal standard and succinylacetone pretreatment solution, adding 100 mu L of the working solution into each hole by using a multi-channel or single-channel liquid-transfering gun in a reverse liquid-adding method, covering a microporous plate with an adhesive sealing film, ensuring good sealing and reducing volatilization; wherein the amino acid isotope internal standard is: 15N 2-13C-Gly、2H4-Ala、2H8-Val、2H3-Leu、2H3-Met、13C6-Phe、13C6-Tyr、2H3-Asp、2H3-Glu、2H2-Orn. The isotopic internal standard of acylcarnitine is:2H9-C0、2H3-C2、2H3-C3、2H3-C4、2H9-C5、2H3-C8、2H9-C14、2H3-C16；
3. placing the microporous plate into an oscillation incubator, incubating and oscillating for 45min at 45 ℃, wherein the rotation speed is 650 plus 750 rpm;
4. taking down the microporous plate after incubation is finished, standing for 5-10min, removing the viscous sealing film, taking care when the film is removed, and preventing the liquid in the plate from overturning;
5. using a multi-channel pipette tip (the pipette tip is moved to one hole and needs to be replaced) to transfer 75 mu L of liquid in the plate to a V-shaped bottom heat-resistant plate;
6. the whole microporous plate is sealed by using an aluminum foil film, and the aluminum foil film is pressed and scratched to be tightly coated, so that the volatilization of liquid is reduced;
7. stabilizing for at least two hours after the transferring step to ensure that succinylacetone in the blood sample is fully derived to obtain a sample loading solution;
8. the microplate was placed into a 2777C autosampler and tested using a Waters TQD instrument, with specific testing procedures as described in the special instructions attached to the instrument.
9. The mass spectrometry results were preliminarily judged according to the reference values shown in table 1. As is clear from Table 1, the phenylalanine content was increased, and the ratio of phenylalanine to other amino acids was abnormal, and therefore, it was judged as hyperphenylalaninemia.
TABLE 1
Second, Gene analysis
1. Washing and dissolving the DNA in the precipitate (in order to increase the concentration, the precipitate in a plurality of plates can be used) in the plate in the first part of step 5 with the solution A, collecting the DNA in an enzyme-removing 1.5mL EP tube, and if necessary, further adding the solution A to the tube to a volume of 200uL, wherein the solution A is an aqueous solution containing 10mM of tris (hydroxymethyl) aminomethane hydrochloride and 1 mM of sodium ethylenediaminetetraacetate, and the pH of the solution A is 8.0;
2. adding 200uL phenol chloroform (1:1) solution and 20 uL premixed solution of proteinase K into the tube, and fully shaking until the color of the blood spot becomes light and white;
3.1200 rpm incubation at 56 deg.C for 1h (or metal bath incubation for 1h, shaking every 15min, instantaneous dissociation);
4. performing instantaneous centrifugation, and standing at room temperature for 5 min;
5. adding 350uL BD solution into the tube, fully reversing and uniformly mixing;
6. transferring all the precipitate and the solution into a CG2 adsorption column, putting the adsorption column into a collection tube, centrifuging at 12000rpm for 30s, discarding liquid in the collection tube, and putting a CG2 adsorption column into the collection tube;
7. adding 500uL GDB solution into the adsorption column, centrifuging at 12000rpm for 30s, discarding liquid in the collection tube, and placing the CG2 adsorption column into the collection tube;
8. adding 600uL of 75% ethanol solution into an adsorption column, centrifuging at 12000rpm for 30s, discarding liquid in a collection tube, and putting a CG2 adsorption column into the collection tube;
9. repeatedly washing once, and putting the CG2 adsorption column into a new collection tube;
10.12000 rpm for 2min, pouring off waste liquid, and air drying the adsorption column at room temperature for 2 min;
11. transferring the adsorption column into a 1.5mL EP tube, suspending and dropwise adding 50 μ L of enzyme-removing deionized water to the middle position of the adsorption film, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, collecting the solution in a centrifuge tube, and measuring the concentration to be 1.2ng/μ L.
12. Library was constructed using the Illumina NGS library preparation kit, the library construction information of which is shown in table 2 and the capture information of which is shown in table 3.
TABLE 2
TABLE 3
| Sampling body Product of large quantities | Quality of | Capture cycle Number of rings | Concentration of trapped product （ng/ul） | Average fraction （bp） | Quality Mean value | qPCR nM | Actual pooling body Product (ul) |
| 6.21 | 98.7 | 10 | 5.76 | 493.2 | 6.77 | 62.01 | 5.81 |
Through second-generation sequencing and comparing a sequencing result with a known whole genome of NCBI, the 158 th G base of the PAH gene is mutated into an A base, the 611 th A base of the PAH gene is mutated into a G base, and both the two positions are pathogenic mutations, so that the mutation of the gene can cause metabolic abnormality, which is consistent with the information of the state of a patient.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
Claims (1)
1. Use of a reagent and an adsorption column for preparing a kit for detecting an object to be detected in a dried blood sample, wherein the reagent is (a) methanol-containing,13C6-a solution of Phe isotope internal standard and succinylacetone, (b) an aqueous solution containing 10mM tris hydrochloride, 1 mM sodium edetate and (c) phenol chloroform; the detection comprises the following steps:
(1) a step of separating a metabolite from a blood spot obtained from said dried blood sample, which comprises using a reagent containing methanol,13C6-oscillating a solution of Phe isotope internal standard and succinylacetone at 40-50 ℃ for 30-60 minutes and separating to obtain a first supernatant and a precipitate, wherein the dried blood sample is a drop of venous blood or peripheral blood collected at a sample collection site and added to a dried blood sample, and the blood spot is a blood spot perforated at the central position of the dried blood spot by a 3.2mm diameter perforator;
(2) treating the precipitate with an aqueous solution containing 10mM tris hydrochloride and 1 mM sodium ethylenediaminetetraacetate, adding phenol-chloroform mixture for further treatment, and centrifuging to obtain a second supernatant;
(3) a step of purifying the genetic material from the second supernatant with an adsorption column to obtain a separated genetic material;
(4) a step of analyzing phenylalanine in the first supernatant by tandem mass spectrometry; and
(5) and (3) constructing a second-generation sequencing library by using the genetic material, wherein the second-generation sequencing result is used for detecting whether the 158 th G base of the PAH gene is mutated into the A base and whether the 611 th A base of the PAH gene is mutated into the G base.
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| KR100742194B1 (en) * | 2005-01-27 | 2007-07-25 | 재단법인서울대학교산학협력재단 | Method for enhancing environmental stress resistance of plant using environmental stress resistance controlling gene |
| US8497065B2 (en) * | 2008-02-15 | 2013-07-30 | Life Technologies Corporation | Methods and kits for extraction of DNA |
| IT1397866B1 (en) * | 2009-12-23 | 2013-02-04 | Azienda Ospedaliero Universitaria Meyer | METHOD AND KIT FOR DETERMINING METABOLITIS ON BLOOD SAMPLES DEPOSITED ON BIBULA PAPER (DRIED BLOOD SPOT). |
| CN108008031A (en) * | 2017-11-17 | 2018-05-08 | 深圳华大生命科学研究院 | Detect amino acid, carnitine, ketone, the method for hormone and kit at the same time |
| CN108051494A (en) * | 2017-11-17 | 2018-05-18 | 深圳华大生命科学研究院 | Method and kit a kind of while that detect a variety of inherited metabolic disease related substanceses |
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