CN107058595B

CN107058595B - Method for detecting schistosome ovum and kit and primer used by same

Info

Publication number: CN107058595B
Application number: CN201710463669.8A
Authority: CN
Inventors: 张洪英; 杨佩才; 谢国祥
Original assignee: NANJING MUNICIPAL CENTER FOR DISEASE CONTROL AND PREVENTION
Current assignee: NANJING MUNICIPAL CENTER FOR DISEASE CONTROL AND PREVENTION
Priority date: 2017-06-19
Filing date: 2017-06-19
Publication date: 2020-05-26
Anticipated expiration: 2037-06-19
Also published as: CN107058595A

Abstract

The invention relates to the field of parasitic disease transmission medium detection, in particular to a schistosome ovum detection method and a kit and primers used by the method. The kit comprises an LAMP primer group, reaction mixed liquor and DNA polymerase; the reaction mixture contains hydroxy naphthol blue as an indicator. The LAMP primer group comprises an outer primer pair consisting of F3 and B3 and an inner primer pair consisting of FIP and BIP; also comprises an LF loop primer. The kit using the detection method can effectively, quickly and specifically detect the schistosome eggs.

Description

Method for detecting schistosome ovum and kit and primer used by same

Technical Field

The invention relates to the field of parasitic disease transmission medium detection, in particular to a schistosome ovum detection method and a kit and primers used by the method.

Background

Schistosomes are also known as Schistosoma japonicum (schistosoma). Schistosomes are parasitic to most vertebrates, and the eggs pass through the vein wall into the bladder and are excreted with the urine. Larvae develop in the intermediate host spiroid. Mature larvae enter the final host through the skin or mouth. Schistosoma mansoni (s. mansoni), is distributed in veins of the large and small intestines, mainly in africa and north south america. The eggs are discharged with the feces. The larvae enter the spiroid and then pass through the skin back into the final host. Schistosoma japonicum (s. japonicum) is mainly found in mainland china, japan, taiwan china, eastern india archipelagic and philippines, and affects other vertebrates such as domestic animals and rats, etc., in addition to humans. The schistosoma japonicum (s. haematobium, i.e. schistosoma japonicum) is mainly distributed in africa, south europe and the middle east. The blood fluke of Egyptian exists in the bladder vein, and the ovum passes through the vein wall and enters the bladder, and is discharged with the urine.

There are many methods for detecting schistosome eggs, and the most common methods in the prior art are as follows: (1) the direct smear method is to smear the excrement of patient or the mucous and bloody excrement of acute schistosome patient directly onto glass slide to prepare excrement film and to identify via microscope based on the morphology of ova, and has simple process and low ova detecting rate. (2) The precipitation method, which is also called egg collection method, is to smear concentrated excrement residue containing worm eggs by a centrifugal precipitation or a standing precipitation or an elutriation method, and the detection rate is improved by microscopic examination compared with the direct smear method. (3) The quantitative transparency method, i.e. the modified Gantteng thickness smear method (Kato-Katz method), is a quantitative method for eggs, i.e. the method for counting eggs in a fixed volume of manure under a microscope. (4) The hatching method of miracidium is characterized by that it utilizes the precipitated egg collected by precipitation method, and makes it culture in chlorine-free warm water (25-30 deg.C), so that the egg can be hatched into miracidium in several hours. The eggs are indirectly detected by observing the metacercaria under a dissecting microscope. (5) Biopsy of rectal mucosa is carried out by taking a small piece of intestinal mucosa from rectum or sigmoid lesion site by proctoscope or sigmoidoscope, tabletting, and observing and identifying ovum under microscope.

However, the existing detection methods have many imperfect places, for example, the most common direct smear method is to detect through morphological distinction identification, the stool has different components in actual operation, and if the method is not an experiential detector, it is sometimes difficult to distinguish and identify fungal spores, different worm eggs and other microorganisms with similar forms, that is, whether the result of the method is accurate or not is highly dependent on the experience of the detector in the morphological distinction identification; the microscopic examination technology has the advantages of simple technology and easy operation, and has the disadvantages of inappropriateness to loose stool and low detection rate, and particularly, the improved rattan-thickened smear method cannot produce satisfactory pieces for the loose stool. In addition, in order to improve the detection rate, operations such as washing, concentrating and hatching the larvae on the feces are often required in actual operation, the time is long, the biological safety requirement of the operation is high, and the basic unit is often operated under the condition without the biological safety requirement, so that the health of operators is directly threatened.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a schistosome ovum detection method and a kit and a primer used by the method, so as to solve the problems.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the LAMP primer group is used for detecting schistosome eggs and comprises an outer primer pair consisting of F3 and B3 and an inner primer pair consisting of FIP and BIP;

the nucleotide sequences of F3, B3, FIP and BIP are sequentially shown as SEQ ID NO 1-4.

Preferably, the LAMP primer group for detecting schistosome eggs further comprises an LF loop primer, and the nucleotide sequence of the LF loop primer is shown as SEQ ID NO. 5.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technology developed in recent years. By designing 4 specific primers for identifying 6 sequences of a target fragment, the whole reaction can be completed within 1h at a constant temperature (60-65 ℃), and the method has the characteristics of sensitivity and specificity compared with the traditional nucleic acid amplification technology due to the adoption of the 4 primers. The reaction does not need precise temperature control equipment and advanced complex analytical instruments, and has low requirements on the proficiency and the professional level of operators, so the method is particularly suitable for quickly detecting the pathogenic microorganisms in a basic level or a laboratory with poor experimental conditions.

The specificity of the primer is the key of success or failure of the LAMP detection method. The design and screening of the primer has unique design principle and rule. The specificity of the reaction is determined by the specificity of the inner primer FIP/BIP and the outer primer F3/B3 in the LAMP reaction.

The primer design of LAMP technology is more complicated than that of PCR technology. It designs 4 specific primers for 6 regions of the target gene. If a pair of loop primers (1oop primers) is added, the reaction can be accelerated, and the amplification efficiency can be improved. The Tm value of the primer is a key factor influencing the normal running of the LAMP reaction, and in a primer group with normal AT/GC content, the Tm values of the F3/B3 primer and the FIP/BIP primer are moderate, so that the amplification efficiency of the primers is optimal AT the temperature.

The loop primer in LAMP accelerates the formation of the loop in the LAMP reaction, and can improve the amplification efficiency and shorten the reaction time.

The Tm values of all primers adopted by the invention accord with the screening principle, and simultaneously, in order to ensure the stability of the tail ends of the primers, the difference between the delta G value of the 3' tail end of the LF/LB and the difference between the delta G value of the 5' end of the F1c and the delta G value of the 5' end of the B1c are less than or equal to-4 Kcal/mol, and F3/B3 and F2/B2 are respectively used for screening the primers. The loop primer LF is located between F1c and F2c, and the terminal delta G value of the LF 3' is less than or equal to-4 Kcal/mol.

The distance between the primers is between 120 and 180bp from the end F25 'to the end B25', the distance between the segment from the 5 'end of F2 to the 3' end of F1c, namely the stem loop, is between 40 and 60bp, and the distance between the 5 'end of F2 to the 3' end of F3 is between 0 and 20 bp. The primer itself cannot form a secondary structure.

A kit for detecting schistosome eggs comprises the LAMP primer group, reaction mixed liquor and DNA polymerase;

preferably, the kit further comprises an indicator; the indicator is selected from one of SYBR Green I, EvaGreen, PicoGreen, Peko Green, propidium iodide, calcein or hydroxy naphthol blue;

more preferably, the indicator is hydroxynaphthol blue, and the concentration of the hydroxynaphthol blue added into the reaction mixed solution is preferably 100 μ M to 140 μ M.

The detection kit has the characteristics of less samples, strong specificity, high sensitivity, rapidness, accuracy and the like, and provides a simple, effective and early rapid diagnosis way for detecting the eggs of the schistosome.

Because the product is double-stranded DNA, the color change can be directly observed, namely, double-stranded DNA fluorescent color reagent such as SYBRGreen I, EvaGreen, PicoGreen, Peko Green, Propidium Iodide (PI) and the like or berberine (berberine) is added into the reaction product, and the color change is observed under a fluorescent lamp, such as Sybr-Green, EvaGreen, PicoGreen, PekoGreen, Green fluorescence in positive and no Green fluorescence in negative. While PI positive is red fluorescence, and PI negative is red fluorescence-free. Berberine has yellow fluorescence in positive fluorescence and no yellow fluorescence in negative fluorescence;

or adding 20-30 mM Calcein and 0.3-0.7 mM MnCl into the reaction system₂Observing the color or fluorescence change during and after the reaction, the negative is orange and the positive is green under visible light, and the color or fluorescence change is observed during and after the reactionNegative under the lamp has no green fluorescence, and positive under the lamp has green fluorescence. Calcein and MnCl₂Color or fluorescence changes can also be observed in the product added after the reaction is complete.

The hydroxynaphthol blue is a metal ion indicator, and is accompanied by Mg in the reaction process²⁺The content reduction gradually changes from light purple to sky blue, and compared with SYBR Green I commonly used in the field, the color change can only occur when effective amplification reaction occurs, so that false positive caused by primer dimer can not occur.

In addition, no additional component is needed for color development of the hydroxynaphthol blue, so that the stability of the reaction system is not interfered. In the observation of the amplification result in the prior art, the result can be observed only by adding a chromogenic reagent after the reaction is finished, and the reaction result cannot be observed in real time; in addition, the PCR tube is opened and a color reagent is added to observe the result, since the concentration of the amplified DNA product is high, up to 10 of the initial concentration⁹And the experimental environment pollution is very easy to cause. The hydroxyl naphthol blue indicator preferably used in the invention has almost no inhibiting effect on a reaction system, can be directly added into the reaction system, can see color change at any time in the reaction process, does not need to observe the result until the reaction is completely finished, and does not need to open a PCR tube.

Preferably, the kit for detecting schistosome eggs comprises the following components:

15 mM-25 mM Tris-HCl with pH of 8.6-9.0, 1.2 mM-1.6 mM dNTPs, 8 mM-12 mM KCl, 8 mM-12 mM (NH)₄)₂SO₄、6mM～10mM MgSO₄0.6-1.0M betaine and 0.07-0.13% Tween 20.

Preferably, the kit for detecting schistosome eggs is the Bst DNA polymerase with strand displacement, and the activity unit is 14-18U/muL.

Bst DNA polymerase is derived from Bacillus stearothermophilus. It has 5'→ 3' polymerase activity and double-strand specific 5'→ 3' exonuclease activity, but no 3'→ 5' exonuclease activity. Has thermal stability, strand displacement activity and polymerase activity, and plays an important role in vitro DNA isothermal amplification reaction.

Preferably, the kit for detecting schistosome eggs as described above further comprises deionized water.

Preferably, the kit for detecting schistosome eggs as described above further comprises a positive control of standard schistosome DNA.

A method for detecting schistosome eggs comprises the following steps:

extracting schistosome ovum DNA to be detected, adding a reagent in the kit of any one of claims 3-5 to form a reaction system, and carrying out a constant temperature reaction; the color change or fluorescence intensity change of the indicator in the reaction solution is observed during or after the reaction.

A large amount of white magnesium pyrophosphate was produced during the LAMP cycle, precipitated at the bottom of the tube and was visible to the naked eye. Therefore, the result can be judged by observing the white precipitate; to compare the amount of reaction product, the turbidity of the reaction product can be detected spectrophotometrically (actually by detecting pyrophosphate produced during the LAMP cycle);

the final product is a mixture of DNA molecules with different lengths, so that similar ladder-shaped bands can be seen by using 2% -3% agarose gel electrophoresis, and positive amplification can also be judged.

Preferably, in the method for detecting schistosome eggs, the molar ratio of F3, B3, FIP, BIP and LF in the reaction system is 1:1 (7-9): 7-9: 3-5;

more preferably, the molar ratio of F3, B3, FIP, BIP and LF is 0.2. mu.M: 1.6. mu.M: 0.8. mu.M.

Preferably, in the method for detecting schistosome eggs, the isothermal reaction conditions are as follows:

reacting at constant temperature of 60-65 ℃ for 60-80 min; more preferably, the reaction is carried out at a constant temperature of 65 ℃ for 66min to 70 min.

Compared with the prior art, the invention has the beneficial effects that:

1) the detection object of the invention is schistosoma japonicum genomic DNA which has the characteristics of conservation and stability, and in addition, 4 primers aiming at 6 sites of the conserved sequence are designed, and the amplification product also has the characteristics of specificity and stability; thus, it is not dependent on the accumulation of morphological experience, and can be grasped by simply learning DNA manipulation.

2) 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'→ 3' polymerization activity, and the specific amplification is realized on the target DNA under the isothermal condition of 60-65 ℃; because the circulation of high-temperature denaturation, low-temperature annealing and extension is not needed, the time required by the conventional PCR temperature change is saved, and the rapid and efficient DNA amplification is realized.

3) The invention reduces the sample exposure as much as possible, protects the sample and the operator to the maximum extent, and has long detection time which only needs 1 hour.

4) The instruments used by the invention are conventional instruments, such as centrifuges, constant-temperature water baths or other constant-temperature equipment, expensive PCR instrument equipment is not needed, and the cost is greatly reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the results of comparative experiments with or without LF primers according to the present invention;

FIG. 2 shows that the primer set provided by the present invention can effectively amplify schistosome ovum DNA, and water negative control can also generate positive reaction after the reaction time is prolonged.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Example 1

Method for extracting schistosome ovum DNA

1. Preparation of stool samples: taking a small amount of fresh collected excrement, about 0.5 g, placing the small amount of fresh collected excrement in a 1.5 ml centrifuge tube, adding 1 ml of physiological saline, fully oscillating on an oscillator, centrifuging for 30 seconds at 14000g, and discarding supernatant; feces stored in alcohol or other solution were washed repeatedly 3 times, and the supernatant was discarded.

2. Cracking: to the pellet was added 200. mu.l of lysis buffer (4M urea, 200mM Tris,20 mM NaCl, 200mM EDTA, pH7.4) followed by 40. mu.l proteinase K (1mg/mL), mixed rapidly and incubated at 55 ℃ for 0.5-1 hour until the solution was clear. The sample was back-and-forth aspirated 3 times with a 1 ml syringe (needle removed).

3. Nucleic acid precipitation: add 200. mu.l binding solution (6M guanidine hydrochloride, 10M urea, 10mM Tris-HCl, 20% Triton X-100(v/v), pH 4.4), mix immediately and incubate at 70 ℃ for 10 min. Add 100. mu.l of isopropanol and mix well.

4. Membrane-adsorbed nucleic acid: transferring all liquid to an adsorption column, centrifuging for 1 minute at 8,000g, and removing the liquid;

5. inhibitor removal: adding 500 μ l inhibitor removing solution (5M guanidine hydrochloride, 20mM Tris-HCl, 40% absolute ethanol (v/v), pH 6.6) into adsorption column, centrifuging at 8,000g for 1 min, and removing the liquid;

6. washing 500. mu.l of washing solution (20mM NaCl, 2mM Tris-HCl, 80% absolute ethanol (v/v), pH 7.5) was added to the adsorption column, and the mixture was centrifuged at 8,000g for 1 minute, and the liquid was discarded. Repeating the steps once;

7. and (3) eluting DNA: 100 μ l of 70 ℃ pre-heated eluate (10mM Tris-HCl, pH 8.5) was added to the adsorption column and centrifuged at 8,000g for 1 minute.

8. And (5) freezing and storing the eluted DNA solution for testing.

Example 2

Loop-mediated isothermal DNA amplification: taking 3 thin-walled tubes for PCR (polymerase chain reaction) with the volume of 0.2mL, and respectively marking the thin-walled tubes as a positive control tube, a detection tube and a negative control tube;

the following ingredients were added to each tube in sequence:

reaction mixture liquid: 15mM Tris-HCl (pH8.6), 1.6mM dNTPs, 8mM KCl, 25mM calcein, 0.5mM MnCl₂、12mM(NH₄)₂SO₄、10mM MgSO₄1.0M betaine (Sigma-Aldrich) and 0.13% by volume percent Tween 20;

16U of Bst DNA polymerase (New England Biolabs);

mixing primers: 0.2 μ M F3 and 0.2 μ M B3, 1.4 μ M FIP and 1.4 μ M BIP and 1.0 μ M LF; the nucleotide sequences of F3, B3, FIP, BIP and LF are sequentially shown in SEQ ID NO. 1-5.

mu.L of the DNA prepared in example 1 was added to the test tube; adding 5 mu L of deionized water into the negative control tube to obtain a negative control; the positive control tube is added with the positive control of the standard schistosome DNA.

Centrifuging for a short time, and reacting in a constant temperature water bath kettle at 63 deg.C for 60 min; bst DNA polymerase was inactivated at 85 ℃ for 2 min. In the reaction process, if target DNA amplification exists, a color reaction can be realized, color change can be observed at any time, the negative color under visible light is orange, the positive color under visible light is green, the negative color under fluorescent lamp does not have green fluorescence, and the positive color under fluorescent lamp is green fluorescence.

Example 3

the following ingredients were added to each tube:

reaction mixture liquid: 25mM Tris-HCl (pH9.0), 1.2mM dNTPs, 8mM KCl, 100. mu.M hydroxynaphthol blue, 8mM (NH)₄)₂SO₄、6mM MgSO₄0.6M betaine (Sigma-Aldrich) and 0.07% by volume Tween 20;

16U of Bst DNA polymerase (New England Biolabs);

mixing primers: 0.2 μ M F3 and 0.2 μ M B3, 1.8 μ M FIP and 1.8 μ M BIP and 0.6 μ M LF; the nucleotide sequences of F3, B3, FIP, BIP and LF are sequentially shown in SEQ ID NO. 1-5.

Centrifuging for a short time, and reacting in a constant temperature water bath kettle at 63 deg.C for 60 min; bst DNA polymerase was inactivated at 85 ℃ for 2 min. In the reaction process, if target DNA is amplified, a color reaction is generated, color change can be observed at any time, a light purple reaction is a negative reaction, and a sky blue reaction is a positive reaction.

Example 4

the following ingredients were added to each tube:

reaction mixture liquid: 20mM Tris-HCl (pH8.8), 1.4mM dNTPs, 10mM KCl, 120. mu.M hydroxynaphthol blue, 10mM (NH)₄)₂SO₄、8mM MgSO₄0.8M betaine (Sigma-Aldrich) and 0.10% by volume Tween 20;

16U of Bst DNA polymerase (New England Biolabs);

mixing primers: 0.2 μ M F3 and 0.2 μ M B3, 1.6 μ M FIP and 1.6 μ M BIP and 0.8 μ M LF; the nucleotide sequences of F3, B3, FIP, BIP and LF are sequentially shown in SEQ ID NO. 1-5.

Experimental example 1

Because the schistosoma japonicum has been evolved for a long time and is highly adaptive to the host, a plurality of genes similar to the host are reserved, and a specific target gene is found as a detection target, which is difficult. To avoid interference from gene families and expressed genes, we selected a non-expressed sequence (GenBank: FN328142.1) as the target.

GenBank:FN328142.1

Thus, the outer primer pair, F3 and B3, as well as the inner forward primer FIP (consisting of F1c and F2), the inner backward primer (consisting of B1c and B2), and the accelerator primer LF were obtained. The orientation of all primers was 5'→ 3'.

A large number of research and application results show that the LAMP method without loop primers is unsuccessful, and the reaction speed is slow; in contrast, the reaction with the addition of the loop primer is greatly accelerated (FIG. 1). FIG. 1 shows that the reaction efficiency is greatly improved after the LF primer is added to the medium copy sample.

(Note: FIG. 1 shows the results of comparative experiments with or without the primers of the present invention, and LF is added; FIG. 1 shows the target DNA is positive control of schistosome ovum DNA. abscissa: time (min); ordinate: FIG. 1-1 shows the relative values calculated by the software, FIG. 1-2 shows the turbidity. obtained by the instrumental detection.)

(Note: FIG. 2 the target DNA is total DNA extracted from different numbers of eggs of schistosome or water negative control. abscissa: time (min); ordinate: turbidity. obtained by instrumental detection.)

Experimental example 2

Study of sensitivity

1) Preparation of standard target DNA molecular template

The purified positive control sample DNA was diluted with TE buffer to 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg, 0.01fg, 0.001 fg.

2) Loop-mediated isothermal DNA amplification and result judgment

Taking 30 thin-walled tubes for PCR (0.2 mL), loading and operating according to the method described in example 4, wherein the difference is that the DNA sequentially added is the standard target DNA molecular template (No. 1-9 tubes) prepared in the previous step, and 5 mu L of deionized water is added into the redundant tube to serve as a negative control (No. 10 tube). 3 replicates per sample

In the reaction process, no difference exists between 1-7 tubes observed under natural light, and sky blue positive reaction can be displayed. Tubes 8, 9 and 10 are light purple. Indicating that the sensitivity of this method is 0.1 fg/. mu.L DNA.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

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Claims

1. The LAMP primer group for detecting the eggs of the schistosome is characterized by comprising an outer primer pair consisting of F3 and B3, an inner primer pair consisting of FIP and BIP and an LF loop primer;

the nucleotide sequences of F3, B3, FIP and BIP are sequentially shown as SEQ ID NO 1-4;

the nucleotide sequence of the LF loop primer is shown as SEQ ID NO. 5.

2. A kit for detecting schistosome eggs, which is characterized by comprising the LAMP primer group, the reaction mixture, the DNA polymerase and the indicator of claim 1;

the indicator is selected from one of propidium iodide, calcein or hydroxynaphthol blue.

3. The kit for detecting schistosome eggs according to claim 2, wherein the indicator is hydroxynaphthol blue, and the concentration of the hydroxynaphthol blue added to the reaction mixture is 100 μ M to 140 μ M.

4. The kit for detecting schistosome eggs according to claim 3, wherein the reaction mixture comprises the following components:

15mM to 25mM Tris-HCl with pH of 8.6 to 9.0, 1.2mM to 1.6mM dNTPs, 8mM to 12mM KCl, 8mM to 12mM (NH)₄)₂SO₄、6mM~10mM MgSO₄0.6-1.0M betaine and 0.07-0.13% Tween20 by volume percent.

5. The kit for detecting schistosome eggs according to claim 3, wherein the DNA polymerase is Bst DNA polymerase with strand displacement, and the activity unit is 14-18U/μ L.

6. The kit for detecting schistosome eggs according to claim 3, wherein the kit further comprises deionized water.

7. The kit for detecting schistosome eggs according to claim 3, wherein the kit further comprises a positive control of standard schistosome DNA.

8. A method for detecting eggs of schistosomes for non-disease diagnostic purposes, comprising:

extracting schistosome ovum DNA to be detected, adding a reagent in the kit of any one of claims 3-5 to form a reaction system, and carrying out a constant temperature reaction; the color change of the indicator in the reaction solution is observed during or after the reaction.

9. The detection method according to claim 8, wherein the molar ratio of F3, B3, FIP, BIP and LF in the reaction system is 1:1 (7-9): (3-5).

10. The detection method according to claim 9, wherein the isothermal reaction conditions are:

reacting at constant temperature of 60-65 ℃ for 60-80 min.