CN116121426B

CN116121426B - A primer set, kit and method for detecting Mycobacterium tuberculosis by loop-mediated isothermal amplification

Info

Publication number: CN116121426B
Application number: CN202310121405.XA
Authority: CN
Inventors: 张洪英; 朱银银
Original assignee: NANJING MUNICIPAL CENTER FOR DISEASE CONTROL AND PREVENTION
Current assignee: NANJING MUNICIPAL CENTER FOR DISEASE CONTROL AND PREVENTION
Priority date: 2023-02-15
Filing date: 2023-02-15
Publication date: 2025-09-23
Anticipated expiration: 2043-02-15
Also published as: CN116121426A

Abstract

The present invention discloses a primer set, a kit, and a method for detecting Mycobacterium tuberculosis by loop-mediated isothermal amplification. The primer set includes an outer primer pair, an inner primer pair, and a loop primer pair; wherein the outer primer pair includes F3 and B3, the nucleotide sequence of F3 is shown in SEQ ID No. 2, and the nucleotide sequence of B3 is shown in SEQ ID No. 3; the inner primer pair includes FIP and BIP, the nucleotide sequence of FIP is shown in SEQ ID No. 4, and the nucleotide sequence of BIP is shown in SEQ ID No. 5; the loop primer pair includes LF and LB, the nucleotide sequence of LF is shown in SEQ ID No. 6, and the nucleotide sequence of LB is shown in SEQ ID No. 7. The detection primers of the present invention use Rv2424c as the target and utilize loop-mediated isothermal amplification detection to rapidly, sensitively, accurately, and efficiently detect Mycobacterium tuberculosis in a sample.

Description

Primer group, kit and method for detecting mycobacterium tuberculosis through loop-mediated isothermal amplification

Technical Field

The invention relates to the technical field of biology, in particular to a primer group, a kit and a method for detecting mycobacterium tuberculosis by loop-mediated isothermal amplification.

Background

According to a report from the world health organization, one third of the world's people are infected with mycobacterium tuberculosis. Infection with this bacterium makes the clinical situation worse for the infected person. Therefore, detection of tubercle bacillus from clinical specimens is very important for treatment and control of infected persons.

One of the conventional methods for detecting Mycobacterium tuberculosis is to detect Mycobacterium tuberculosis by morphological distinction and identification, and in practice, sputum has various components, and if it is not an experienced person, it is sometimes difficult to prepare sputum chips suitable for detection and stain them. After acid-fast staining of sputum smears, it was observed under a microscope that Brevibacterium rubrum was positive, but staining positivity only indicated the presence of acid-fast bacilli, and was indistinguishable from other acid-fast staining positives, especially from tubercle bacilli and nontuberculous mycobacteria. Another conventional detection method is a separation culture method, which has a higher sensitivity than a smear microscopy method, but a culture period of up to 6-8 weeks. Conventional detection methods are also molecular biological detection methods, which include Polymerase Chain Reaction (PCR), isothermal amplification (SAT, LAMP, RPA, RAA, etc.), molecular Hybridization (MH), etc., which are more sensitive and greatly reduce the time required for detection (from several hours to one day). Molecular detection methods can also detect, identify strains directly from specimens and isolated culture in the laboratory. However, the continuous denaturation, renaturation and extension are needed, the cyclic reciprocation is needed, the temperature is needed to be continuously adjusted, and the whole reaction time is prolonged by nearly one time in the temperature adjustment process. Moreover, the detection of different targets of Mycobacterium tuberculosis has been disclosed, including groEl, mtb-4 and dnaJ genes encoding 32kDa, 38kDa and 65kDa antigens, insertion sequences, 16S-23S spacer heat shock protein (hsp) 65 encoding genes and 16S rRNA. IS986 and IS6110 are the most common repeat elements in most mycobacterium tuberculosis, with 10-16 copies in the strain. Among them, IS6110 IS more sensitive and more specific than IS 986. IS6110 IS considered as a useful target for detection of mycobacterium tuberculosis. However, the presence of this target in mycobacterium bovis can lead to false positive results. Furthermore, it has been reported that some Mycobacterium tuberculosis strains lack such components, which may lead to false negative results.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a primer group, a kit and a method for detecting mycobacterium tuberculosis, wherein the detection primer takes Rv2424c as a target, and the mycobacterium tuberculosis in a sample can be detected rapidly, sensitively, accurately and efficiently by utilizing a loop-mediated isothermal amplification detection technology.

The target gene Rv2424c used in the present invention is a specific transposase of a recently discovered Mycobacterium tuberculosis insertion sequence, which is commonly found in Mycobacterium tuberculosis group.

Transposases, which are enzymes that perform a transposable function, are usually encoded by a transposon, recognize specific sequences at both ends of the transposon, and are capable of detaching the transposon from adjacent sequences and inserting it into a new DNA target site. In bacteria, because transposases of the insertion sequences differ in their catalytically active central amino acid sequences, their transposases are classified as DDE transposases, DEDD transposases, HUH transposases and serine transposases. Insertion of an insertion sequence into the coding region of a gene by a different transposable mechanism results in mutation, deletion and inversion of the gene, or insertion upstream of the gene, affects expression of the gene downstream of the insertion sequence by either its own promoter or by forming a hybrid promoter with the gene, thereby helping the bacterium resist complex environmental changes. To date, at least 2000 insert sequences were found. In mycobacteria, the first insertion was found in the 80 s of the 20 th century. To date, at least 37 different mycobacterial insert sequences have been found.

Mycobacterium tuberculosis IS an acid-tolerant and host-restricted aerobic pathogen, carrying about 30 different insertion sequence elements, the most studied of which IS6110, which IS present only in the Mycobacterium tuberculosis complex (MTBC) members. At present, foreign scholars use PCR to amplify Rv2424c in order to study the function of transposase, and no detection method using the PCR amplified Rv2424c as a DNA detection target exists. The invention adopts the Rv2424c gene as a mycobacterium tuberculosis isothermal amplification detection target, and provides a high-sensitivity and rapid detection method of an MTBC new target Rv2424 c.

The invention is realized in the following way:

in a first aspect, the invention provides a primer set for detecting mycobacterium tuberculosis by loop-mediated isothermal amplification, wherein the primer set takes Rv2424c as a target gene and comprises an outer primer pair, an inner primer pair and a loop primer pair;

wherein, the outer primer pair comprises F3 and B3, the nucleotide sequence of F3 is shown as SEQ ID No.2, and the nucleotide sequence of B3 is shown as SEQ ID No. 3;

The inner primer pair comprises FIP and BIP, the nucleotide sequence of the FIP is shown as SEQ ID No.4, and the nucleotide sequence of the BIP is shown as SEQ ID No. 5;

The loop primer pair comprises LF and LB, the nucleotide sequence of the LF is shown as SEQ ID No.6, and the nucleotide sequence of the LB is shown as SEQ ID No. 7.

In a second aspect, the invention also provides a kit for detecting mycobacterium tuberculosis by loop-mediated isothermal amplification, which comprises the LAMP primer group.

The addition amount and the proportion relation of the primers can influence the amplification result, and in order to obtain the amplification result which is easier to distinguish, the molar ratio of the outer primer pair to the inner primer pair to the loop primer pair is preferably 1:8:4.

In some embodiments, the above-described kits further comprise one or more of dNTPs, PCR buffer, and Bst DNA polymerase.

The Bst DNA polymerase is used for replacing the DNA polymerase of the conventional PCR, the Bst DNA polymerase has high temperature resistance, a strand displacement function and DNA 5 '. Fwdarw.3' polymerization activity, and can realize specific amplification of target DNA under the isothermal condition of 60-65 ℃, and the circulation of high temperature denaturation, low temperature annealing and extension is not needed, so that the time required by the conventional PCR temperature change is saved, and the rapid and efficient DNA amplification is realized.

In some embodiments, the PCR buffer comprises Tris-HCl, mnCl ₂,(NH₄)₂SO₄,MgSO₄, tween 20, betaine, and an indicator.

The indicator in the kit is selected from a DNA indicator or a metal ion indicator. The invention is not limited by the use of DNA indicator or metal ion indicator, wherein the DNA indicator refers to fluorescent indicator and can be Syto 9, and the metal ion indicator can be calcein or hydroxy naphthol blue.

In a third aspect, the present invention provides a method for detecting mycobacterium tuberculosis, which comprises performing loop-mediated isothermal amplification reaction on a sample to be detected by using the primer set or the kit, and determining the result by color change after the amplification reaction is finished.

The reaction process of the loop-mediated isothermal amplification reaction is shown in FIG. 1:

The FIP primer hybridizes to the F2 region of the target DNA to synthesize a complementary strand, the LAMP reaction starts (FIG. 1B), bst DNA polymerase (with strand displacement function) displaces the complementary strand synthesized by the FIP primer with the extended strand of the external primer (F3 or B3) (FIG. 1C), the complementary strand synthesized by the FIP primer is released and becomes a template for the BIP primer (FIG. 1C), and a dumbbell-shaped DNA is formed (FIG. 1D) which enters the LAMP cycle as a template (FIG. 1D). LF is used as another primer and enters the circular amplification until the reaction is finished. The final product is a mixture of DNA molecules (stem-loop DNAs) of different lengths having a strand-loop structure.

In the present invention, the above-mentioned 25. Mu.L reaction system for the amplification reaction was 10mM Tris-HCl (pH=7.1), 50mM KCl, 10mM MnCl ₂, 10mM indicator, 8mM MgSO ₄, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 2% glycerol, 0.8M betaine, 0.2. Mu.M outer primer pair, 1.6. Mu.M inner primer pair, 0.8. Mu.M ring primer pair, 1.4mM dNTPs, 8U/. Mu.L Bst DNA polymerase and 5. Mu.L DNA sample.

As shown in fig. 2 and 3, when the indicator is calcein, the color shows yellowish color, shows positive color, shows pale pink color, shows negative color, shows green fluorescence, shows positive color, shows no fluorescence, and shows negative color under ultraviolet light.

The result of the invention can be presented by a plurality of methods, is convenient and efficient, 1 is that the color change is observed by visible light, 2 is that whether green fluorescence exists is observed under an ultraviolet lamp, 3 is that the fluorescence value can be detected in real time in a fluorescence instrument with a FAM/SYBGREEN channel and can be interpreted by an amplification curve and a Ct value, and 4 is that the instrument with a dissolution curve mode can be interpreted by an annealing temperature and a curve.

When the indicator is Syto 9, the color shows green fluorescence under ultraviolet light, indicating positive, and the color shows no fluorescence, indicating negative.

When the indicator is hydroxynaphthol blue, the color shows sky blue, positive, light purple and negative under visible light.

In some embodiments, the loop-mediated isothermal amplification is performed under conditions of 62-68℃for 20-40min and 80-99℃for 1-3min to inactivate Bst DNA polymerase. Preferably, the reaction conditions are 65℃for 30min and 80-99℃for 2min to inactivate Bst DNA polymerase.

The primer group, the reaction system and the reaction conditions are adopted for loop-mediated isothermal amplification, so that the method not only has the characteristics of good stability and high sensitivity, but also can be used for rapidly detecting and obtaining an amplification curve in a short time, and compared with the conventional PCR amplification reaction which takes at least 2 hours, the method only takes 10 minutes to 30 minutes.

In a fourth aspect, the invention also provides application of the primer group, the kit or the method in detection of mycobacterium tuberculosis.

The invention has the following beneficial effects:

(1) Compared with smear microscopy, the invention can specifically detect the tubercle bacillus genes without amplifying genes of other organisms by detecting the genome in the tubercle bacillus, has no dependence on morphology, and can distinguish other acid-staining positive bacteria from nontuberculous mycobacteria by amplifying the Rv2424c gene which is specific to the tubercle bacillus complex as a target gene, and has strict specificity.

(2) The invention aims at the specific Rv2424c gene of the mycobacterium tuberculosis, which is used as a target gene for amplification, and the gene only exists in the mycobacterium tuberculosis complex, so that the mycobacterium tuberculosis complex which causes human infection can be identified, and the specificity is high without amplifying non-tuberculosis mycobacterium.

(3) Compared with the conventional PCR method, the isothermal amplification technology is adopted, the temperature does not need to be regulated, the reaction time is shortened to one fourth, the original PCR amplification reaction needs at least 2 hours, and the isothermal amplification technology only needs 10 minutes to 30 minutes.

(4) The result of the invention can be presented by various methods, is convenient and efficient, and is suitable for various instruments and scenes.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a reaction process of a loop-mediated isothermal amplification reaction according to the present invention;

FIG. 2 shows the detection result under visible light in example 1 of the present invention, wherein the left side is a negative control, the middle is a positive sample, and the right side is a positive control;

FIG. 3 shows the detection result under ultraviolet light in example 1 of the present invention, wherein the left side is a negative control, the middle is a positive sample, and the right side is a positive control;

FIG. 4 shows the positions of each primer of the Rtb145 primer set corresponding to the Rv2424c template;

FIG. 5 shows the result of Rtb145 sensitivity detection in example 3 (A：H37Ra:4x10⁴CFU/ml;B：H37Ra:4x10³CFU/ml;C：H37Ra:4x10²CFU/ml;D：H37Ra:4x10⁰CFU/ml).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The present embodiment is a method for detecting Mycobacterium tuberculosis, comprising:

extraction of DNA:

(1) Sample treatment, namely taking a sputum specimen, adding 1-2 times of volume of sputum (containing 2% NaOH) into the sputum specimen according to the nature, adding 0.5% N-acetyl-L-cysteine before using, fully and uniformly mixing, and standing at room temperature for 10-15 minutes. 1-3 ml 13000g of the mixture was centrifuged at room temperature for 15 minutes, and the supernatant was discarded.

(2) Cleavage, adding 10 microliter proteinase K (1 mg/mL) into the precipitate, mixing rapidly, incubating for 10 minutes at 100 ℃, and cooling for 10 minutes in a refrigerator at-20 ℃.

(3) Nucleic acid purification

① The nucleic acid precipitate was suspended by adding 300. Mu.l of the adsorption solution and immediately mixing.

② The column was inserted into a2 ml blank tube.

③ Membrane adsorbing nucleic acid by transferring all the liquid to adsorption column, centrifuging at 13,000g for 1 min, and discarding penetrating liquid;

④ Washing the column, adding 700 microliter of washing solution (80% absolute ethanol (v/v)) into the column, centrifuging for 1 minute at 13,000g, and discarding the penetration solution. Repeating the process once;

⑤ Drying nucleic acid by placing the empty adsorption column in a centrifuge and centrifuging at 13,000g for 2 minutes.

⑥ Eluting DNA, namely sleeving an adsorption column into a new 1.5 ml centrifuge tube, adding 60 microliters of eluent into the adsorption column, and centrifuging for 1 minute at 13,000 g.

(4) Preserving nucleic acid, and detecting DNA liquid eluted by freezing at-20 ℃.

2. Primer set design and screening:

(1) Primers were designed based on the gene sequence of Mycobacterium tuberculosis Nc_000962.3:2720776-272177Rv 2424c [ organosm= Mycobacterium tuberculosis H37Rv, wherein the nucleotide sequence of Rv2424c is shown as SEQ ID No. 1. The designed LAMP specific primer group capable of identifying mycobacterium tuberculosis is named as Rtb145, and the position corresponding to the template Rv2424c is shown in FIG. 4. The primer set Rtb145 comprises two outer primers (F3 and B3), two inner primers (FIP consisting of the complementary sequences of F1c and F2; BIP consisting of B1c and B2) and two loop primers (LF and LB), the sequences of which are shown in Table 1.

TABLE 1 primer sequences of primer set Rtb145

	Sequence(s)	SEQ ID No.
			F3	AGGTCCGCAAGTTCGGT	2
B3	TCCTTGGGCTTCGAGTTTG	3
			FIP(F1c+F2)	AGCACGTGCCAAATGATGACGACAGTCCCGCTGCCAAC	4
BIP(B1c+B2)	GCCTCACCAGGATCTCGGCCGTTCTTTGTCGGGATCCAT	5
			LF	TCAGCTTGTGGGCGACG	6
LB	GCCGACTACTTCACCACCC	7

3. Loop-mediated isothermal amplification reaction

And (3) adding the sample DNA in the step (1) into a reaction system, and placing the reaction system in an amplification instrument to perform an amplification reaction. Wherein 25. Mu.L of the reaction system comprises 10mM tris–HCl(pH 7.1),50mM KCl,10mM MnCl₂,10mM Calcein(Sigma–Aldrich),8mM MgSO₄,0.1mM EDTA,1mM DTT,0.1％ Triton X-100,2％ glycerol, 0.8M betaine (Sigma-Aldrich), 0.2. Mu.M F3 and B3, 1.6. Mu.M FIP and BIP, 0.8. Mu.M LF and LB,1.4mM dNTPs,8units Bst DNA polymerase and 5. Mu.L of sample DNA.

The reaction condition of the amplification reaction is that the reaction is carried out for 30min at 65 ℃ and then the Bst DNA polymerase is inactivated for 2min at 80-99 ℃.

In the detection, three groups of tests including water-negative control, positive control of tubercle bacillus (H37 a) DNA and positive sample are provided. The experimental results are shown in fig. 2 and 3, wherein the left side is negative control, the middle is positive sample, and the right side is positive control. The negative control and the negative sample are light pink under visible light and have no fluorescence under an ultraviolet lamp, and the positive control and the positive sample are light yellow under visible light and have green fluorescence under the ultraviolet lamp.

Example 2

This example is a verification of the effect of the primer set provided in example 1

1. TB-DNA as template

The Rtb145 primer set was amplified at 65℃for 30 minutes, the water control had no amplification curve, the amplification curve appeared after the addition of TB-DNA, the amplification curve was obtained for an average time of 8 minutes and 22 seconds, and the annealing temperature was 91.46 ℃. The results are shown in Table 2:

TABLE 2 Rtb145 primer set screening results

2. The cross-reactivity with Mycobacterium bovis was examined using gradient diluted BCG as a template and the results are shown in Table 3. The results indicate that primer set Rtb145 was amplified for 30 minutes and was reactive against 250CFU/ML BCG bacteria.

TABLE 3 Cross-reactivity results with BCG

3. Clinical sample is used as template

The detection results of clinical samples show that the Rtb145 can rapidly detect 10 samples to obtain an amplification curve, wherein the time average is 8 minutes and 18 seconds, the shortest time is 5 minutes and 45 seconds, and the longest time is 12 minutes. The annealing temperature was 91.46 ℃ on average and the results are shown in table 4.

TABLE 4 detection results of primer set Rtb145 on 10 samples

Example 3

Sensitivity detection of LAMP primer group of example 1

The results of isothermal amplification with the primer set Rtb145 after gradient dilution of the inactivated Mycobacterium tuberculosis H Ra were shown in FIG. 5, wherein A：H37Ra:4x10⁴CFU/ml;B:H37Ra:4x10³CFU/ml;C：H37Ra:4x10²CFU/ml;D：H37Ra:4x10⁰CFU/ml. shows that the Rtb145 primer set was effective for amplification of TB H37Ra 4X 10 ² CFU/ML.

Example 4

Specific detection of LAMP primer group of example 1

Isothermal amplification with primer set Rtb145 was not responsive to Mycobacterium avium, mycobacterium terrestris, mycobacterium schneider, mycobacterium kansasii, mycobacterium asiaticum, mycobacterium scrofula, mycobacterium gordonae, mycobacterium torticola, mycobacterium fortuitum, mycobacterium grass, influenza virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, coxsackie virus, adenovirus, nocardia brazil, corynebacterium beijing, pneumococcus, legionella pneumophila, botrytis cinerea, mycoplasma pneumoniae, haemophilus and staphylococci.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer set for detecting Mycobacterium tuberculosis by loop-mediated isothermal amplification, characterized in that the primer set comprises an outer primer pair, an inner primer pair, and a loop primer pair;

The outer primer pair includes F3 and B3, the nucleotide sequence of F3 is shown in SEQ ID No. 2, and the nucleotide sequence of B3 is shown in SEQ ID No. 3;

The inner primer pair includes FIP and BIP, the nucleotide sequence of FIP is shown in SEQ ID No. 4, and the nucleotide sequence of BIP is shown in SEQ ID No. 5;

The loop primer pair includes LF and LB, the nucleotide sequence of LF is shown in SEQ ID No.6, and the nucleotide sequence of LB is shown in SEQ ID No.7.

2. A kit for detecting Mycobacterium tuberculosis by loop-mediated isothermal amplification, characterized in that it comprises a LAMP reaction solution containing the primer set according to claim 1.

3. The kit according to claim 2, wherein the molar ratio of the outer primer pair, the inner primer pair, and the loop primer pair in the LAMP reaction solution is 1:8:4.

4. test kit according to claim 3, is characterized in that, described test kit also comprises one or more in dNTPs, PCR buffer and BstDNA polymerase.

The kit according to claim 4 , wherein the PCR buffer comprises: Tris-HCl, MnCl ₂ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ , Tween 20, betaine and an indicator.

The kit according to claim 5 , wherein the indicator is selected from a DNA indicator or a metal ion indicator.

7. The kit according to claim 6, wherein the DNA indicator is Syto 9.

8. The kit according to claim 6, wherein the metal ion indicator is hydroxynaphthol blue and calcein.

9. A method for detecting Mycobacterium tuberculosis, characterized in that the method is for non-disease diagnosis purposes, comprising performing a loop-mediated isothermal amplification reaction on a test sample using the primer set of claim 1 or the kit of any one of claims 2 to 8, and determining the result by color change after the amplification reaction is completed.

10. The method according to claim 9, characterized in that the reaction system for loop-mediated isothermal amplification is 25 μL: 10 mM tris-HCl (pH 7.1), 50 mM KCl, 10 mM MnCl ₂ , 10 mM indicator, 8 mM MgSO ₄ , 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 2% glycerol, 0.8 M betaine, 0.2 μM F3 and B3, 1.6 μM FIP and BIP, 0.8 μM LF and LB, 1.4 mM dNTPs, 8 units Bst DNA polymerase and 5 μL sample DNA.

11 . The method according to claim 10 , wherein the reaction conditions of the loop-mediated isothermal amplification are: reaction at 62-68° C. for 20-40 min, and then inactivation of Bst DNA polymerase at 80-99° C. for 1-3 min.