CN113684245A

CN113684245A - Detection kit and detection method for MPT64 protein

Info

Publication number: CN113684245A
Application number: CN202010423197.5A
Authority: CN
Inventors: 尹小毛
Original assignee: Fifth Affiliated Hospital Of Southern Medical University
Current assignee: Fifth Affiliated Hospital Of Southern Medical University
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2021-11-23

Abstract

The invention relates to a detection kit and a detection method of MPT64 protein, wherein the detection kit comprises a first antibody, a second antibody, template DNA, liposome raw materials and an amplification primer pair; wherein the first antibody and the second antibody are capable of specifically binding to MPT64 protein and the amplification primer pair is capable of specifically amplifying the template DNA. The detection method of the invention uses the first antibody to directly coat the PCR reaction tube, replaces an enzyme label plate form in the immunoliposome PCR method, and establishes the rapid hypersensitivity detection method of the MPT64 protein based on the improved immunoliposome technology, thereby improving the simplicity of immunoliposome PCR and further improving the sensitivity of protein detection.

Description

Detection kit and detection method for MPT64 protein

Technical Field

The invention relates to the technical field of biological detection, in particular to a detection kit and a detection method for MPT64 protein.

Background

Although tuberculosis is effectively controlled worldwide, there is a long distance from the goal of eliminating tuberculosis. In recent years, the aggravation of global population movement, the rise of AIDS combined tuberculosis infection cases, the increase of multi-drug resistant and pan-drug resistant tuberculosis bring great challenges to tuberculosis prevention and treatment. Meanwhile, the development of the tuberculosis diagnosis and treatment field, particularly the early diagnosis is very small, and the existing various tuberculosis rapid diagnosis technologies cannot simultaneously meet the requirements of simplicity, accuracy and low cost. In the face of severe tuberculosis prevention and treatment situation, development and application of new rapid diagnosis methods have become the core content of tuberculosis clinical research. At present, laboratory diagnosis of Tubercle Bacillus (TB) mainly depends on traditional smear and culture methods, but the methods are complex to operate and long in detection period, and the requirements of clinical TB rapid diagnosis cannot be met. Some rapid diagnostic methods developed in recent years, such as real-time fluorescence PCR and cell immunoassay, have solved the problem of diagnostic time, but are not satisfactory in terms of diagnostic accuracy.

The MPT64 protein is a group of proteins secreted and released outside the bacteria body and dissociated in a culture medium in the early and middle culture periods of the mycobacterium tuberculosis, is coded by RD2 region gene Rv1980c, has the relative molecular mass of 24000 and accounts for about 8 percent of the total amount of the proteins in a supernatant filter solution of a mycobacterium tuberculosis culture medium. Therefore, the MPT64 protein can be used as a target protein for rapid diagnosis of Mycobacterium tuberculosis.

The immunoliposome PCR technology is a new protein hypersensitivity detection method which is established and gradually developed in recent years, and the application prospect of the immunoliposome PCR technology in the medical field, particularly in the aspect of disease diagnosis, is very promising. The basic principle of detecting target protein is as follows: firstly, coating an enzyme-labeled plate hole with an anti-target protein antibody, then adding a sample to be detected, incubating for a certain time, adding a biotinylated anti-target protein antibody, then respectively adding avidin and biotinylated liposome, washing the plate after reacting for a period of time to remove free biotinylated liposome, destroying the liposome by using a membrane breaking agent to release a DNA fragment coated by the liposome into a solution, absorbing a small amount of the solution to perform Taqman real-time fluorescence PCR, and amplifying the Ct value to reflect the concentration of the target protein in the sample to be detected. The defects in the prior art are that the prior immunoliposome PCR technology can not realize single-tube detection, and the whole operation process is complex and tedious.

Disclosure of Invention

Therefore, a detection kit for the MPT64 protein, which can realize single-tube detection and is fast and convenient to operate, is needed.

A detection kit for MPT64 protein comprises a first antibody, a second antibody, template DNA, liposome raw materials and an amplification primer pair; wherein the first antibody and the second antibody are capable of specifically binding to MPT64 protein and the amplification primer pair is capable of specifically amplifying the template DNA.

In one embodiment, the sequence of the template DNA is as set forth in SEQ ID NO: 1 is shown.

In one embodiment, the sequences of the amplification primer pairs are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

In one embodiment, the kit further comprises PCR buffer, a fluorescent probe and DNA polymerase.

In one embodiment, the liposome starting material comprises cholesterol, lecithin, and phosphatidylethanolamine.

In one embodiment, diethyl ether, chloroform, glutaraldehyde and BSA are also included.

In one embodiment, the first antibody is a murine antibody and the second antibody is a rabbit antibody.

The invention also provides a detection method of MPT64 protein for non-disease diagnosis and treatment purposes, which comprises the following steps:

coating the PCR reaction tube with a first antibody capable of being specifically combined with the MPT64 protein to obtain a coated reaction tube;

labeling a liposome wrapped with template DNA with a second antibody capable of specifically binding to MPT64 protein to obtain immunoliposome;

adding a sample to be detected into the coating reaction tube for incubation, and then adding the immunoliposome for incubation;

washing the coated reaction tube by using a cleaning buffer solution, and then adding a PCR reaction solution to amplify the template DNA;

detecting the content of the amplification product, and calculating the content of the MPT64 protein in the sample to be detected according to the content of the amplification product.

In one embodiment, the liposome is prepared from cholesterol, lecithin and phosphatidylethanolamine.

In one embodiment, the molar ratio of cholesterol, lecithin and phosphatidylethanolamine is (9-11): (5-7): 1-3.

The principle of the MPT64 protein detection kit is as follows:

the PCR reaction tube is coated by the first antibody, and the liposome coating the template DNA is labeled by the second antibody to prepare the immunoliposome. And adding a sample to be detected into the coated PCR reaction tube, incubating for a period of time, and then adding immunoliposome, so that the MPT64 protein in the sample to be detected can form a first antibody-MPT 64 protein-second antibody immunoliposome compound with the first antibody and the second antibody in sequence. Then removing the liquid in the PCR reaction tube, adding a buffer solution for washing, and adding the PCR reaction liquid to amplify the template DNA released by the liposome. The amount of the amplified product is in direct proportion to the amount of the template DNA in a fixed time, and the amount of the template DNA is in direct proportion to the MPT64 protein content in the sample to be detected, so that the MPT64 protein content in the sample to be detected can be calculated by detecting the content of the amplified product.

Compared with the existing detection kit and detection method, the invention has the following beneficial effects:

(1) the operation is simple and convenient: in the test process, the plate washing is not required to be carried out on the ELISA plate for many times, and the buffer solution is directly added into the PCR reaction tube for washing, so that the single-tube detection can be realized.

(2) High specificity: the MPT64 protein is specifically detected by adopting a double-antibody sandwich mode, and the reaction is less influenced by high-abundance protein in serum.

(3) High sensitivity: the lowest value detected can reach fg/mL, which can be as low as 1/2000 of ELISA method.

Drawings

FIG. 1 is a transmission electron micrograph of the immunoliposome prepared in example 2.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The kit for detecting the MPT64 protein, provided by the embodiment of the invention, comprises a first antibody, a second antibody, template DNA, a liposome raw material and an amplification primer pair. Wherein the first antibody and the second antibody are capable of specifically binding to MPT64 protein, and the pair of amplification primers are capable of specifically amplifying the template DNA.

The principle of the MPT64 protein detection kit is as follows:

The term "antibody" refers to a class of immunoglobulins that specifically bind to an antigen. An antigen is usually composed of multiple epitopes, one epitope stimulates the body, and an antibody produced by a B lymphocyte receiving the antigen is called a monoclonal antibody. Stimulation of the body by multiple epitopes correspondingly produces a wide variety of monoclonal antibodies, which when mixed together are polyclonal antibodies, and the antibodies produced in the body are polyclonal antibodies.

The term "liposome" refers to a closed ring vesicle composed of lipid bilayers that is non-toxic and non-immunogenic. The liposome is classified according to the structure and the particle size, and comprises unilamellar liposome, multichamber liposome, multiphase liposome and the like. Unilamellar liposomes, i.e., liposomes whose contents are encapsulated by only one lipid bilayer, are divided into small unilamellar liposomes and large unilamellar liposomes depending on their diameter. Multilamellar liposomes, also known as multilamellar liposomes, are heterogeneous aggregates formed by the inclusion of several lipid bilayers. By multi-phasic liposomes is meant multi-phasic dispersions based on unilamellar or multilamellar liposomes comprising a small amount of a water-in-oil or oil-in-water emulsion.

The template DNA is an artificially designed DNA fragment, and may be a single-stranded molecule, a double-stranded molecule, a linear molecule, or a circular molecule. In one specific example, the sequence of the template DNA is as set forth in SEQ ID NO: 1 is shown. It is to be understood that the above is a preferred example, and the template DNA is not limited thereto and may be designed as required.

SEQ ID NO：1：5’-TCGAGGCGTAGAATTCCCTTACCGATGCGCGCTG TCTACTCTGACAATTGCATATGTGGTCTATGTCGTCGTTCGCTAGTAGTTCC TGGGCTGCAC-3’

In one specific example, the sequences of the amplification primer pairs are set forth in SEQ ID NOs: 2 and SEQ ID NO: 3, respectively. It is understood that the above is a preferred example and the amplification effect is good, but the amplification primer set is not limited thereto and may be designed as required.

SEQ ID NO: 2 (upstream primer): 5'-TCGAGGCGTAGAATTCCCT-3'

SEQ ID NO: 3 (downstream primer): 5'-GTGCAGCCCAGGAACTACT-3'

In one particular example, the detection kit further comprises a PCR buffer, a fluorescent probe, and a DNA polymerase. Optionally, the sequence of the fluorescent probe is as set forth in SEQ ID NO: 4, respectively.

SEQ ID NO: 4(Taqman probe):

5’-FAM-TGTCTACTCTGACAATTGCAT-BQ1-3’

optionally, the PCR buffer solution contains 10 mmol/L-30 mmol/L Tris-HCl, 0.5 mmol/L-5 mmol/L MgCl₂50 to 80mmol/L of (NH)₄)₂SO₄And KCl with the concentration of 40 mmol/L-60 mmol/L, the DNA polymerase is Bst DNA polymerase.

In one particular example, the liposome starting material includes cholesterol, lecithin, and phosphatidylethanolamine. Alternatively, the liposome encapsulating the template DNA may be prepared by: dissolving cholesterol, lecithin and phosphatidylethanolamine in a mixed solvent of diethyl ether and chloroform, and evaporating under reduced pressure on a rotary evaporator to form a film. Then, ether, chloroform mixture and Tris-HCl buffer solution of DNA fragment (artificial sequence) were added, and sonication was performed until the mixed solution was hydrated. And then rotationally evaporating the mixed solution in a water bath at 45 ℃ to form a viscous gel, evaporating the gel under reduced pressure to form a phase inversion to a liquid state, supplementing a certain amount of Tris-HCl buffer solution, and performing ultrasonic treatment to obtain the liposome wrapped with the template DNA fragment.

In one specific example, the detection kit further comprises diethyl ether, chloroform, glutaraldehyde and BSA to facilitate preparation of liposomes encapsulating the template DNA fragment and labeling of the secondary antibody. Alternatively, the method of labeling the liposome entrapped with the template DNA fragment with the second antibody is as follows: mixing glutaraldehyde solution with liposome solution coated with template DNA fragment, slightly shaking at 30 deg.C for 2 hr, and dialyzing at 4 deg.C with PBS solution for 12 hr to remove excess glutaraldehyde. Adding a second antibody into the dialyzed solution, standing at 4 ℃ for 12 hours, adding an equal volume of BSA solution, and standing for 12 hours to form the immunoliposome. The immunoliposome solution is centrifuged at 20000r/min at 4 ℃ for 30min, and after the supernatant is discarded, the washing is repeated 1 time with PBS buffer. Finally, the immunoliposome was resuspended in 3mL of PBS buffer and stored at 4 ℃ for further use.

In one specific example, the first antibody is a murine antibody and the second antibody is a rabbit antibody. It is to be understood that the type of antibody is not limited thereto, and may be selected as desired.

The method for detecting the MPT64 protein, provided by the embodiment of the invention, comprises the following steps of S1-S5:

s1, coating the PCR reaction tube with a first antibody capable of specifically binding with the MPT64 protein to obtain a coated reaction tube.

S2, labeling the liposome wrapped with the template DNA with a second antibody capable of specifically binding with the MPT64 protein to obtain the immunoliposome.

And S3, adding the sample to be detected into the coated reaction tube for incubation, and then adding the immunoliposome for incubation.

S4, washing the coated reaction tube by using a washing buffer solution, and adding a PCR reaction solution to amplify the template DNA.

S5, detecting the content of the amplification product, and calculating the content of the MPT64 protein in the sample to be detected according to the content of the amplification product.

The detection method of the invention uses the first antibody to directly coat the PCR reaction tube, replaces an enzyme label plate form in the immunoliposome PCR method, and establishes the rapid hypersensitivity detection method of the MPT64 protein based on the improved immunoliposome technology, thereby improving the simplicity of immunoliposome PCR and further improving the sensitivity of protein detection. The detection method can be used for accurately detecting the trace MPT64 protein in samples such as bacteria culture solution, blood, cerebrospinal fluid, pleural effusion and the like. It will be appreciated that the detection method may also be used for non-disease diagnostic and therapeutic purposes, for example to detect the presence of MPT64 protein in food or in the environment, to determine if the food or environment is contaminated with mycobacterium tuberculosis, etc.

Compared with the existing detection method, the invention has the following beneficial effects:

In one particular example, liposomes are prepared from cholesterol, lecithin, and phosphatidylethanolamine. Specifically, cholesterol, lecithin and phosphatidylethanolamine are dissolved in a mixed solvent of diethyl ether and chloroform, and the mixture is evaporated on a rotary evaporator under reduced pressure to form a film. Then, ether, chloroform mixture and Tris-HCl buffer solution of DNA fragment (artificial sequence) were added, and sonication was performed until the mixed solution was hydrated. And then rotationally evaporating the mixed solution in a water bath at 45 ℃ to form a viscous gel, evaporating the gel under reduced pressure to form a phase inversion to a liquid state, supplementing a certain amount of Tris-HCl buffer solution, and performing ultrasonic treatment to obtain the liposome wrapped with the template DNA fragment. In one embodiment, the molar ratio of cholesterol, lecithin and phosphatidylethanolamine is (9-11): (5-7): 1-3.

In one specific example, the reaction conditions for amplification are: at 95 ℃ for 30 s; 40 cycles of 95 ℃, 30s, 60 ℃ and 30 s.

In one specific example, a method of coating a PCR reaction tube includes the steps of: adding the first antibody into a PCR reaction tube, standing for 12h, adding a BSA solution, standing for 12h, removing liquid in the tube, and drying at room temperature.

In one specific example, the method of labeling liposomes with a second antibody comprises the steps of: the glutaraldehyde solution was mixed with the liposome solution coated with template DNA, gently shaken at 30 ℃ for 2 hours, and then dialyzed at 4 ℃ against PBS solution for 12 hours to remove excess glutaraldehyde. Adding a second antibody into the dialyzed solution, standing at 4 ℃ for 12 hours, adding an equal volume of BSA solution, and standing for 12 hours to form the immunoliposome. The immunoliposome solution is centrifuged at 20000r/min at 4 ℃ for 30min, and after the supernatant is discarded, the washing is repeated 1 time with PBS buffer. Finally, the immunoliposome was resuspended in 3mL of PBS buffer and stored at 4 ℃ for further use.

In one specific example, the detection method comprises the following steps:

(1) formation of PCR reaction tube coated antibody-MPT 64 protein complex: placing a sample to be detected (blood serum, cerebrospinal fluid and the like) in the coated PCR reaction tube, and standing at room temperature for 30min to form a coated antibody-MPT 64 protein complex;

(2) formation of coated antibody-MPT 64 protein-immunoliposome complexes: adding the immunoliposome compound into the liquid obtained in the step (1), gently mixing uniformly, and standing at room temperature for 30min to form a coating antibody-MPT 64 protein-immunoliposome compound;

(3) removing redundant immunoliposomes: and (3) removing the redundant liquid in the PCR reaction tube in the step (2), adding a buffer solution, and standing for 5 min. Repeating the steps;

(4) heating and breaking the film: adding PCR reaction solution into the PCR reaction tube, heating at 100 ℃ for 10min, and releasing the wrapped template DNA after the liposome is damaged;

(5) and (3) PCR detection: and (3) placing the PCR reaction tube into a fluorescent quantitative PCR instrument for detection, and determining the content of the MPT64 protein in the sample by analyzing the content of the template DNA.

The following are specific examples.

Example 1: coated PCR reaction tube

mu.L of mouse anti-MPT 64 antibody (here anti-MPT 64 antibody 1, diluted with carbonate buffer) was added to a PCR reaction tube, left at 4 ℃ for 12 hours, 150. mu.L of BSA solution was added thereto, left at 12 hours, the liquid in the tube was removed, dried at room temperature, and stored at 4 ℃ for further use.

Example 2: preparation of immunoliposomes

1. Cholesterol, lecithin and phosphatidylethanolamine are mixed according to a molar ratio of 10: 6: 2 is dissolved in a mixed solvent of ether and chloroform, and is evaporated on a rotary evaporator under reduced pressure to form a film. Then, ether, chloroform mixture and Tris-HCl buffer solution of DNA fragment (template DNA) were added thereto, and sonication was carried out until the mixed solution was hydrated. And then rotationally evaporating the mixed solution in a water bath at 45 ℃ to form a viscous gel, evaporating the gel under reduced pressure to form a phase inversion to a liquid state, supplementing a certain amount of Tris-HCl buffer solution, and performing ultrasonic treatment to obtain the liposome wrapped with the DNA fragment.

2. 2mL of glutaraldehyde solution was mixed with 2mL of liposome solution coated with DNA fragment, gently shaken at 30 ℃ for 2 hours, and then dialyzed at 4 ℃ against PBS solution for 12 hours to remove excess glutaraldehyde. 1mL of rabbit anti-MPT 64 antibody (here anti-MPT 64 antibody 2) is added into the dialyzed solution, the solution is placed for 12 hours at 4 ℃, and then an equal volume of BSA solution is added for 12 hours to form the immunoliposome.

3. The immunoliposome solution is centrifuged at 20000r/min at 4 ℃ for 30min, and after the supernatant is discarded, the washing is repeated 1 time with PBS buffer. Finally, the immunoliposome was resuspended in 3mL of PBS buffer and stored at 4 ℃ for further use. The prepared immunoliposome can be observed by a transmission electron microscope, as shown in figure 1.

Example 3: construction of PCR System

1. The PCR detection amplified template DNA is an artificially designed DNA fragment, a primer group comprises an upstream primer and a downstream primer and a Taqman probe, and the specific sequence is as follows:

target gene:

5’-TCGAGGCGTAGAATTCCCTTACCGATGCGCGCTGTCTACTCTGACAA TTGCATATGTGGTCTATGTCGTCGTTCGCTAGTAGTTCCTGGGCTGCAC-3’；

an upstream primer: 5'-TCGAGGCGTAGAATTCCCT-3', respectively;

a downstream primer: 5'-GTGCAGCCCAGGAACTACT-3', respectively;

taqman probe: 5 '-FAM-TGTCTACTCTGACAATTGCAT-BQ 1-3'.

2. Adopting a Taqman probe method fluorescent quantitative PCR detection mode, wherein the amplification conditions are as follows: at 95 ℃ for 30 s; 40 cycles of 95 ℃, 30s, 60 ℃ and 30 s.

3. The PCR detection result is judged by analyzing the real-time fluorescence detection data of the fluorescence quantitative PCR instrument, and the content of the protein to be detected is determined by making a standard curve.

Example 4: detection of MPT64 protein in human serum samples

1. Formation of first antibody-MPT 64 protein complex: 50 mu L of serum sample to be detected is put into a PCR reaction tube coated with the first antibody, and the first antibody-MPT 64 protein complex can be formed after the first antibody-MPT 64 protein complex is placed for 30min at room temperature.

2. Formation of first antibody-MPT 64 protein-immunoliposome complexes: adding 50 μ L of immunoliposome complex of anti-MPT 64 protein, mixing, and standing at room temperature for 30 min.

3. Washing: sucking away the liquid in the tube by using a sample adding gun, and adding 100 mu L of PBS buffer solution for washing once; this step is repeated.

4. Heating and breaking the film: adding 20 mu L of PCR reaction solution, wherein the reaction system is shown in the following table 1; heating at 100 deg.C for 10min to destroy liposome and release coated template DNA (artificial sequence).

5. And (3) PCR detection: the reaction condition is 95 ℃ for 30 s; 40 cycles of 95 ℃ for 30s and 60 ℃ for 30 s. The PCR detection result is judged by analyzing real-time fluorescence detection data of the fluorescence quantitative PCR instrument, and the content of MPT64 protein in the sample is determined by making a standard curve.

TABLE 1 PCR reaction System

Component name	Addition amount (μ L)	Component name	Addition amount (μ L)
				Buffer solution	10	Bst DNA polymerase	0.8
Upstream primer	4	Fluorescent dyes	0.4
				Downstream primer	0.5
Taqman probe	2	Adding water to make up to	20

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> fifth subsidiary hospital of southern medical university

<120> MPT64 protein detection kit and detection method

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 96

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tcgaggcgta gaattccctt accgatgcgc gctgtctact ctgacaattg catatgtggt 60

ctatgtcgtc gttcgctagt agttcctggg ctgcac 96

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcgaggcgta gaattccct 19

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtgcagccca ggaactact 19

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtctactct gacaattgca t 21

Claims

1. The kit for detecting the MPT64 protein is characterized by comprising a first antibody, a second antibody, template DNA, a liposome raw material and an amplification primer pair; wherein the first antibody and the second antibody are capable of specifically binding to MPT64 protein and the amplification primer pair is capable of specifically amplifying the template DNA.

2. The detection kit of claim 1, wherein the sequence of the template DNA is as set forth in SEQ ID NO: 1 is shown.

3. The detection kit according to claim 2, wherein the sequences of the amplification primer pair are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

4. The detection kit of claim 1, further comprising a PCR buffer, a fluorescent probe, and a DNA polymerase.

5. The test kit of claim 1, wherein the liposome starting material comprises cholesterol, lecithin, and phosphatidylethanolamine.

6. The test kit of claim 1, further comprising diethyl ether, chloroform, glutaraldehyde, and BSA.

7. The detection kit according to claim 1, wherein the first antibody is a murine antibody and the second antibody is a rabbit antibody.

8. A method for detecting MPT64 protein for non-disease diagnostic and therapeutic purposes, comprising the steps of:

washing the coated reaction tube by using a washing buffer solution, and then amplifying the template DNA;

9. The method of claim 8, wherein the liposome is prepared from cholesterol, lecithin, and phosphatidylethanolamine.

10. The detection method according to claim 9, wherein the molar ratio of cholesterol, lecithin and phosphatidylethanolamine is (9-11): (5-7): (1-3).