CN112063736A - Rapid plasmodium detection kit and detection method based on loop-mediated isothermal amplification technology - Google Patents

Abstract

The invention relates to the technical field of plasmodium detection, in particular to a plasmodium rapid detection kit and a plasmodium rapid detection method based on a loop-mediated isothermal amplification technology. The kit for rapidly detecting the plasmodium based on the loop-mediated isothermal amplification technology comprises a plasmodium falciparum detection primer group reagent, a vivax malaria detection primer group reagent, a plasmodium malariae detection primer group reagent, an oval malaria detection primer group reagent, LAMP amplification reaction liquid, DNA polymerase and a color developing agent; the LAMP amplification reaction solution comprises buffer solution and dNTPs; the buffer solution contains magnesium ions and does not contain a metal chelating agent; the color developing agent comprises calcein and MnCl₂. The invention can quickly and accurately identify four plasmodium species of plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale.

Description

Plasmodium rapid detection kit and detection based on loop-mediated isothermal amplification technology method

technical field

The invention relates to the technical field of Plasmodium detection, in particular to a rapid detection kit and detection method of Plasmodium based on loop-mediated isothermal amplification technology.

Background technique

Malaria is an insect-borne infectious disease that threatens human health and life safety, and is listed as one of the three major public health problems in the world. (P.m) and 4 species of Plasmodium ovale (P.o). At present, the detection methods of Plasmodium in China mainly include microscopy and fluorescent PCR.

Microscopic examination is a routine method. Although the microscopic examination is intuitive, it has obvious defects. It is not sensitive to low malaria parasite and mixed infection. There are few malaria cases, the skills of microscopy personnel are becoming increasingly unfamiliar, and the malaria microscopy capability has a tendency to degrade. The WHO external assessment of China's Plasmodium microscopy capabilities, negative blood, Plasmodium falciparum, and Plasmodium vivax blood were scored as follows: 92.5%, 78.3%, 70.8%, the missed diagnosis and misdiagnosis were higher by microscopy.

PCR can accurately identify Plasmodium species, but it is expensive and can only be carried out in a well-equipped laboratory. The experimental time is long, which limits its application in primary hospitals and areas with relatively poor medical conditions.

In addition, foreign detection methods for Plasmodium also include P. falciparum (P.f) and other Plasmodium mixed LAMP method detection kits, which cannot distinguish between Plasmodium vivax (P.v), Plasmodium vivax (P.m), and Plasmodium ovale (P.o)3 Plasmodium species.

SUMMARY OF THE INVENTION

In order to solve the above problems, the purpose of the present invention is to provide a rapid detection kit and detection method for Plasmodium based on loop-mediated isothermal amplification technology, which can rapidly, accurately and accurately identify Plasmodium falciparum, P. Four Plasmodium species of Plasmodium form.

In order to achieve the above object, the rapid detection kit for Plasmodium based on the loop-mediated isothermal amplification technology designed in the present invention is characterized in that it includes a primer set reagent for detection of falciparum malaria, a primer set reagent for detection of vivax malaria, and a set of primer set reagents for detection of malaria vivax. Primer set reagent, ovale detection primer set reagent, LAMP amplification reaction solution, DNA polymerase, chromogenic reagent;

The falciparum malaria detection primer set includes: Pf-FIP: SEQ ID NO1, Pf-BIP: SEQ ID NO2, Pf-F3: SEQ ID NO3, Pf-B3: SEQ ID NO4;

The primer set for detection of vivax malaria includes: Pv-FIP: SEQ ID NO7, Pv-BIP: SEQ ID NO8, Pv-F3: SEQ ID NO9, Pv-B3: SEQ ID NO10;

The three-day malaria detection primer set includes: Pm-FIP: SEQ ID NO13, Pm-BIP: SEQ ID NO14, Pm-F3: SEQ ID NO15, Pm-B3: SEQ ID NO16;

The primer set for detection of Malaria ovale includes: Po-FIP: SEQ ID NO19, Po-BIP: SEQ ID NO20, Po-F3: SEQ ID NO21, Po-B3: SEQ ID NO22;

The LAMP amplification reaction solution includes a buffer and dNTPs; the buffer contains magnesium ions and does not contain a metal chelator;

The color developer includes calcein and MnCl ₂ .

As a preferred solution, the primer set reagents for detection of malaria falciparum also include: Pf-LF: SEQ ID NO5, Pf-LB: SEQ ID NO6; the primer set for detection of vivax malaria further includes Pv-LF: SEQ ID NO11, Pv -LB: SEQ ID NO12; the primer set for detection of Plasmodium vivax also includes Pm-LF: SEQ ID NO17, Pm-LB: SEQ ID NO18; the primer set for detection of P. ovale also includes: Po-LF: SEQ ID NO23, Po-LB: SEQ ID NO24.

As a preferred solution, the primer set reagents for detection of falciparum malaria include: 2.67 μmol/L Pf-FIP, 2.67 μmol/L Pf-BIP, 0.33 μmol/L Pf-F3, 0.33 μmol/L Pf-B3, 0.67 μmol/L Pf-LF , 0.67μmol/L Pf-LB; vivax malaria detection primer set reagents include: 2.67μmol/L Pv-FIP, 2.67μmol/L Pv-BIP, 0.33μmol/L Pv-F3, 0.33μmol/L Pv-B3, 0.67μmol/L Pv-LF, 0.67μmol/L Pv-LB; primer set reagents for the detection of malaria malaria include: 2.67μmol/L Pm-FIP, 2.67μmol/L Pm-BIP, 0.33μmol/L Pm-F3, 0.33 μmol/L Pm-B3, 0.67 μmol/L Pm-LF, 0.67 μmol/L Pm-LB; primer set reagents for detection of Plasmodium ovale include: 2.67 μmol/L Po-FIP, 2.67 μmol/L Po-BIP, 0.33 μmol /L Po-F3, 0.33μmol/L Po-B3, 0.67μmol/L Po-LF, 0.67μmol/LPo-LB; LAMP amplification reaction solution includes: 33.3mmol/L Tris-HCl, 16.7mmol/L KCl, 16.7 mmol/L (NH ₄ ) ₂ SO ₄ , 13.3 mmol/L MgSO ₄ , 0.1% Triton-X100, 2.3 mmol/L dNTPs, the pH of the LAMP amplification reaction solution was 8.8; the enzymatic activity of Bst DNA polymerase was 8000U/ml; the developer includes 0.65mmol/L calcein, 7.8mmol/LMnCl ₂ , 50% dimethyl sulfoxide.

As a preferred solution, it also includes a DNA extraction solution, which includes 30% H ₂ O ₂ , 0.1 mol/L NaOH, and 1 mol/L Tris-HCl at pH 8.0.

A rapid detection method for Plasmodium based on loop-mediated isothermal amplification technology, comprising the steps of:

(1) Extract the DNA of the sample to be tested;

(2) Loop-mediated isothermal amplification:

(2.1) Prepare the detection system of falciparum malaria, vivax malaria detection system, three-day malaria detection system and ovale malaria detection system respectively:

According to the volume ratio, the LAMP amplification reaction solution: falciparum malaria detection primer set: Bst DNA polymerase: chromogenic reagent is 15:6:1:1 to prepare the falciparum malaria detection system;

According to the volume ratio, LAMP amplification reaction solution: vivax malaria detection primer set: Bst DNA polymerase: chromogenic reagent 15:6:1:1 to prepare the vivax malaria detection system;

According to the volume ratio, the LAMP amplification reaction solution: Plasmodium spp. detection primer set: Bst DNA polymerase: chromogenic reagent is 15:6:1:1 to prepare the Pseudomonas spp. detection system;

According to the volume ratio, LAMP amplification reaction solution: Plasmodium ovale detection primer set: Bst DNA polymerase: chromogenic reagent 15:6:1:1 to prepare a detection system for Plasmodium ovale;

(2.2) Place the detection system of falciparum malaria, vivax malaria detection system, triscal malaria detection system and ovale malaria detection system in four PCR tubes respectively, and add the DNA of the sample to be tested to each PCR tube, 60 React at ~65°C for 45-90 minutes; observe the color of the liquid in each PCR tube, green fluorescence is positive, and orange is negative.

As a preferred solution, take 1-5 μl of peripheral blood or drop 1-5 μl of dried blood on filter paper into a centrifuge tube, add 10 μl of 30% H ₂ O ₂ and 50 μl of 0.1 mol/L NaOH, lyse at 95°C for 20 min, and cool to room temperature Add 50 μl of 1 mol/L Tris-HCl at pH 8.0 and mix well to obtain the DNA of the sample.

As a preferred solution, step (2) is specifically as follows: three groups of parallel experiments are set up in each of the falciparum malaria detection system, the vivax malaria detection system, the three-day malaria detection system and the ovale malaria detection system, and one group is the experimental group. , the DNA of the sample to be tested is added to the system, the other group is the negative control group, the DNA of healthy human blood is added to the system, the last group is the blank control group, and deionized water is added to the system; 60-65 ℃ reaction 45 to 90 minutes; observe the color of the liquid in each PCR tube separately.

The principle of the invention is as follows: the kit designs four primers for the six regions on the target sequence of the malaria parasite, namely F3, B3, FIP and BIP, and designs the loop primers LF and LB in order to improve the reaction speed, and designs four groups for the four malaria parasites. Different species-specific primers. The 3' and 5' ends of primers in FIP and BIP recognize two different regions in the target nucleic acid sequence, respectively, and are designed so that their 5' end sequences anneal and bind to the complementary chain region synthesized by the extension reaction initiated by the 3' end. The inner primers of FIP and BIP can form a stem-loop structure and carry out self-extension reaction, and then a new inner primer is annealed and combined at the position of the loop structure and undergoes a strand displacement synthesis reaction, and the cycle is repeated to realize the amplification reaction. Under the action of Bst DNA polymerase, dNTPs are converted to generate pyrophosphate, which reacts with magnesium ions in the solution to generate a large amount of magnesium pyrophosphate precipitates. A metal ion color developer is added to the reaction solution. When the nucleic acid is used, calcein will automatically bind to the double-stranded DNA, and it will show different colors due to the different content of magnesium ions in the reaction solution. If an amplification reaction occurs, the reaction solution will turn green. If no amplification reaction occurs , the color of the reaction solution is orange.

The advantages of the present invention are: compared with the existing Plasmodium detection method, the Plasmodium rapid detection kit based on the loop-mediated isothermal amplification technology of the present invention can identify the target by designing LAMP primer sets of four types of Plasmodium. The nucleic acid sequence is amplified under the action of Bst DNA polymerase, and the positive and negative can be judged by the naked eye through the chromogenic agent, which can quickly, accurately and accurately identify falciparum malaria, vivax malaria, Triscal malaria, and ovale malaria.

Description of drawings

Fig. 1 is a graph of the test results of using the kit of the present invention to specifically detect samples of Plasmodium falciparum, Plasmodium vivax, Plasmodium vivax, and Plasmodium ovale. Plasmodium vivax human blood sample, tube 3 is a human blood sample with P. vivax, tube 4 is a human blood sample with P. ovale, tube 5 is a healthy human blood sample, tube 6 is a human blood sample with Leishmania, Tube No. 7 is a human blood sample with Schistosoma japonicum, and tube No. 8 is a blank (H ₂ O) sample;

Fig. 2 is the ring-mediated isothermal amplification efficiency diagram of Plasmodium falciparum; wherein the dotted line is the turbidity peak curve without adding the color developer, and the solid line is the turbidity peak curve with the color developer added;

Fig. 3 is the loop-mediated isothermal amplification efficiency diagram of Plasmodium vivax; wherein the dotted line is the turbidity peak curve without adding the color developer, and the solid line is the turbidity peak curve with the color developer added;

Fig. 4 is the loop-mediated isothermal amplification efficiency diagram of Plasmodium malariae; wherein the dotted line is the turbidity peak curve without adding the chromogenic agent, and the solid line is the turbidity peaking curve with the chromogenic agent added;

Figure 5 is a diagram of the efficiency of ring-mediated isothermal amplification of Plasmodium ovale; the dotted line is the turbidity peak curve without the addition of the color developer, and the solid line is the turbidity peak curve with the addition of the color developer.

Detailed ways

For a better understanding of the present invention, the invention will be described in detail below with reference to specific examples.

Example 1 Plasmodium rapid detection kit and detection method for Plasmodium based on loop-mediated isothermal amplification technology of the present invention

The rapid detection kit for Plasmodium based on loop-mediated isothermal amplification technology in this embodiment includes DNA extraction solution, primer set reagents for detection of Plasmodium falciparum, primer set reagents for detection of vivax malaria, primer set reagents for detection of Plasmodium ovale, Detection primer set reagent, LAMP amplification reaction solution, DNA polymerase, chromogenic reagent;

DNA extraction solution includes 30% H ₂ O ₂ , 0.1 mol/L NaOH, 1 mol/L Tris-HCl at pH 8.0;

Plasmodium falciparum detection primer set reagents include: 2.67μmol/L Pf-FIP (SEQ ID NO1), 2.67μmol/L Pf-BIP (SEQ ID NO2), 0.33μmol/L Pf-F3 (SEQ ID NO3), 0.33μmol/ L Pf-B3 (SEQ ID NO4), 0.67 μmol/L Pf-LF (SEQ ID NO5), 0.67 μmol/L Pf-LB (SEQ ID NO6);

vivax malaria detection primer set reagents include: 2.67μmol/L Pv-FIP (SEQ ID NO7), 2.67μmol/L Pv-BIP (SEQ ID NO8), 0.33μmol/L Pv-F3 (SEQ ID NO9), 0.33μmol /L Pv-B3 (SEQ ID NO10), 0.67 μmol/L Pv-LF (SEQ ID NO11), 0.67 μmol/L Pv-LB (SEQ ID NO12);

The primer set reagents for the detection of malaria malaria include: 2.67 μmol/L Pm-FIP (SEQ ID NO13), 2.67 μmol/LPm-BIP (SEQ ID NO14), 0.33 μmol/L Pm-F3 (SEQ ID NO15), 0.33 μmol/ L Pm-B3 (SEQ ID NO16), 0.67 μmol/L Pm-LF (SEQ ID NO17), 0.67 μmol/L Pm-LB (SEQ ID NO18);

Plasmodium ovale detection primer set reagents include: 2.67μmol/L Po-FIP (SEQ ID NO19), 2.67μmol/LPo-BIP (SEQ ID NO20), 0.33μmol/L Po-F3 (SEQ ID NO21), 0.33μmol/ L Po-B3 (SEQ ID NO22), 0.67 μmol/L Po-LF (SEQ ID NO23), 0.67 μmol/L Po-LB (SEQ ID NO24);

The LAMP amplification reaction solution includes: 33.3 mmol/L Tris-HCl, 16.7 mmol/L KCl, 16.7 mmol/L (NH ₄ ) ₂ SO ₄ , 13.3 mmol/L MgSO ₄ , 0.1% Triton-X100, 2.3 mmol/L dNTPs, the pH of LAMP amplification reaction solution is 8.8;

The enzymatic activity of Bst DNA polymerase is 8000U/ml;

The developer includes 0.65 mmol/L calcein, 7.8 mmol/L MnCl ₂ , and 50% dimethyl sulfoxide.

The method for detecting malaria parasite using the kit of the present embodiment includes the steps:

(1) Extract the DNA of the sample to be tested

Take the filter paper with 1-5 μl blood droplets adsorbed into a centrifuge tube, add 10 μl of 30% H ₂ O ₂ and 50 μl of 0.1 mol/L NaOH, lyse at 95°C for 20 min, cool to room temperature and add 50 μl of 1 mol/L pH8 .0 Tris-HCl, and mix to obtain the DNA of the sample.

(2) Loop-mediated isothermal amplification:

(2.1) Preparation of the detection system of falciparum malaria, vivax malaria detection system, triticaria malaria detection system and ovale malaria detection system:

Take 15 μl of LAMP amplification reaction solution, 6 μl of falciparum detection primer set, 1 μl of Bst DNA polymerase, and 1 μl of chromogenic reagent to prepare a falciparum malaria detection system;

Take 15 μl of LAMP amplification reaction solution, 6 μl of vivax malaria detection primer set, 1 μl of Bst DNA polymerase, and 1 μl of chromogenic reagent to prepare a vivax malaria detection system;

Take 15 μl of LAMP amplification reaction solution, 6 μl of the primer set for detection of Malaria malaria, 1 μl of Bst DNA polymerase, and 1 μl of chromogenic reagent to prepare the detection system of Malaria malaria;

Take 15 μl of LAMP amplification reaction solution, 6 μl of oval malaria detection primer set, 1 μl of Bst DNA polymerase, and 1 μl of chromogenic reagent to prepare a detection system for ovale malaria;

Take 12 PCR tubes and number them as 1, 2, and up to 12. The falciparum malaria detection system is placed in PCR tubes 1 to 3, and the vivax malaria detection system is placed in PCR tubes 4 to 6; 7 to 6 Tube No. 9 is respectively loaded into the detection system for Malaria ovale; tubes No. 10 to 12 are respectively loaded into the detection system for Plasmodium ovale.

(2.2) Add 2 μl of the DNA of the sample to be tested to 12 PCR tubes respectively, and react at 65°C for 60 minutes; observe the color of the liquid in each PCR tube, green fluorescence is positive, and orange is negative.

Embodiment 2 Accuracy of the detection method of Plasmodium of the present invention

16 samples of P. falciparum (P.f), 16 samples of P. vivax (P.v), 4 samples of P. vivax (P.m), 16 samples of P. ovale (P.o) and 12 samples of healthy human DNA were subjected to conventional The results of the detection methods of microscopy and PCR are consistent with the method of the present invention using the kit, indicating that the detection method of the protozoa of the present invention has high accuracy.

Example 3 Sensitivity of the detection method for Plasmodium of the present invention

As shown in Table 1, the detection method (hereinafter referred to as LAMP) of the Plasmodium rapid detection kit based on the loop-mediated isothermal amplification technology of the present invention, as well as microscopic examination and PCR detection of 120 malaria cases, all were positive, and χ ² were respectively. The Kappa values were 0.961 and 0.987, respectively, with high consistency.

Table 1

Example 4 Specificity of the detection method for Plasmodium of the present invention

With reference to Fig. 1, tubes 1 to 4 are green, and the detection is positive, tubes 5 to 8 are orange, and no positive is detected, and 10 samples of Leishmania are detected by the method for detecting malaria parasite of the present invention, 8 samples of Schistosoma japonicum and 48 samples of healthy human blood were all in orange, and none of them were positive, indicating that the detection method for Plasmodium of the present invention has strong specificity.

Example 5 Sensitivity of the detection method for Plasmodium of the present invention

Take 25000 Pf/μl blood, 3000 Pv/μl blood, 2000 Pm/μl blood, 4000 Po/μl blood to extract 5μl of DNA and dilute them into 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^{- 4.} From stock solution to 10 ^-4 DNA dilution solution, follow LAMP operation, from stock solution to 10 ^-3 DNA dilution solution, the LAMP of 4 species of Plasmodium all turned green fluorescence, and 10 ^-4 DNA dilution solution of 4 species of Plasmodium showed no color change. Calculated, the sensitivity of Pf, Pv, Pm, Po was 25, 3, 2, and 4 Plasmodium/μl blood, respectively.

Example 6 Reproducibility of the detection method for Plasmodium of the present invention

4 samples of Pf, Pv, Pm, Po, 4 parallel samples respectively, all LAMP positive, 1 healthy and 1 blank (H ₂ O) sample, 4 parallel samples, all negative.

Example 7 Loop-mediated isothermal amplification efficiency of the detection method for Plasmodium of the present invention

Combined with Table 2 and Figure 2, Figure 3, Figure 4, Figure 5, LAMP was measured by the LA-500 real-time turbidity detection system, and the turbidity peak time of 4 kinds of Plasmodium LAMP without chromogenic agent and with chromogenic agent added Between 20-32min and 26-40min, the peak turbidity rate was between 0.14-0.17 and 0.1-0.13, respectively, and the four species of Plasmodium all had high amplification efficiency.

Table 2

Example 8 Reagent storage time of the rapid detection kit for Plasmodium based on loop-mediated isothermal amplification technology of the present invention

After storing the reagents in the kit for 1 month, 2 months and 6 months, 8 Pf, 8 Pv, 5 Pm, 8 Po, 8 healthy and 3 blank (H ₂ O) samples were detected, and the results All match. It is shown that the storage time of each reagent in the kit of the present invention can reach more than this year.

To sum up, the rapid detection method for Plasmodium based on the loop-mediated isothermal amplification technology of the present invention has the following advantages compared with the current detection kits, microscopy and PCR detection methods:

(1) The present invention can simultaneously detect and identify 4 species of Plasmodium (P. falciparum, P. vivax, P. vivax, P. ovale), and the current detection kit cannot distinguish between P. vivax (P.v) and P. vivax (P. vivax). (P.m), Plasmodium ovale (P.o) 3 species of Plasmodium.

(2) The cost of the present invention is low, the detection time is short, and the rate of missed diagnosis and misdiagnosis is low. Compared with PCR detection, the cost of the reagent of the present invention is less than one tenth of that of the market Plasmodium fluorescent PCR method, and the detection time is 10 times less than that of the fluorescent PCR method. One-half, compared with the method of microscopic inspection, greatly reduces the missed detection rate.

(3) The present invention has the characteristics of high accuracy, good specificity and high sensitivity, and is suitable for large-scale production and application.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

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Claims (7)

1. a rapid detection kit for Plasmodium based on loop-mediated isothermal amplification technology, is characterized in that, comprises the detection primer set reagent of Plasmodium falciparum, the detection primer set reagent of vivax malaria, the detection primer set reagent of Malaria var. Malaria detection primer set reagent, LAMP amplification reaction solution, DNA polymerase, chromogenic reagent; The falciparum malaria detection primer set includes: Pf-FIP: SEQ ID NO1, Pf-BIP: SEQ ID NO2, Pf-F3: SEQ ID NO3, Pf-B3: SEQ ID NO4; The primer set for detection of vivax malaria includes: Pv-FIP: SEQ ID NO7, Pv-BIP: SEQ ID NO8, Pv-F3: SEQ ID NO9, Pv-B3: SEQ ID NO10; The three-day malaria detection primer set includes: Pm-FIP: SEQ ID NO13, Pm-BIP: SEQ ID NO14, Pm-F3: SEQ ID NO15, Pm-B3: SEQ ID NO16; The primer set for detection of Plasmodium ovale includes: Po-FIP: SEQ ID NO19, Po-BIP: SEQ ID NO20, Po-F3: SEQ ID NO21, Po-B3: SEQ ID NO22; The LAMP amplification reaction solution includes a buffer and dNTPs; the buffer contains magnesium ions and does not contain a metal chelator; The color developer includes calcein and MnCl ₂ . 2. The rapid detection kit for Plasmodium based on loop-mediated isothermal amplification technology according to claim 1, characterized in that, the detection primer set reagent for Plasmodium falciparum further comprises: Pf-LF: SEQ ID NO5, Pf- LB: SEQ ID NO6; the vivax malaria detection primer set further includes Pv-LF: SEQ ID NO11, Pv-LB: SEQ ID NO12; the three vivax malaria detection primer set further includes Pm-LF: SEQ ID NO17, Pm-LB: SEQ ID NO18; the described P. ovale detection primer set further includes: Po-LF: SEQ ID NO23, Po-LB: SEQ ID NO24. 3. the rapid detection kit of Plasmodium based on loop-mediated isothermal amplification technology according to claim 2, is characterized in that, Falciparum malaria detection primer set reagents include: 2.67μmol/L Pf-FIP, 2.67μmol/L Pf-BIP, 0.33μmol/LPf-F3, 0.33μmol/L Pf-B3, 0.67μmol/L Pf-LF, 0.67μmol/ L Pf-LB; vivax malaria detection primer set reagents include: 2.67μmol/L Pv-FIP, 2.67μmol/L Pv-BIP, 0.33μmol/LPv-F3, 0.33μmol/L Pv-B3, 0.67μmol/L Pv-LF, 0.67μmol /L Pv-LB; The primer set reagents for the detection of malaria malaria include: 2.67μmol/L Pm-FIP, 2.67μmol/L Pm-BIP, 0.33μmol/LPm-F3, 0.33μmol/L Pm-B3, 0.67μmol/L Pm-LF, 0.67μmol /L Pm-LB; Plasmodium ovale detection primer set reagents include: 2.67μmol/L Po-FIP, 2.67μmol/L Po-BIP, 0.33μmol/LPo-F3, 0.33μmol/L Po-B3, 0.67μmol/L Po-LF, 0.67μmol /L Po-LB; The LAMP amplification reaction solution includes: 33.3 mmol/L Tris-HCl, 16.7 mmol/L KCl, 16.7 mmol/L (NH ₄ ) ₂ SO ₄ , 13.3 mmol/L MgSO ₄ , 0.1% Triton-X100, 2.3 mmol/L dNTPs, the pH of LAMP amplification reaction solution is 8.8; The enzymatic activity of Bst DNA polymerase is 8000U/ml; The developer includes 0.65 mmol/L calcein, 7.8 mmol/L MnCl ₂ , and 50% dimethyl sulfoxide. 4. The rapid detection kit for Plasmodium based on loop-mediated isothermal amplification technology according to claim 1, further comprising a DNA extraction solution comprising 30% H ₂ O ₂ , 0.1 mol/L NaOH , 1 mol/L Tris-HCl at pH 8.0. 5. A rapid detection method for Plasmodium based on loop-mediated isothermal amplification technology according to any one of claims 1 to 4, characterized in that, comprising the steps of: (1) Extract the DNA of the sample to be tested; (2) Loop-mediated isothermal amplification: (2.1) Prepare the detection system of falciparum malaria, vivax malaria detection system, three-day malaria detection system and ovale malaria detection system respectively: According to the volume ratio, the LAMP amplification reaction solution: falciparum malaria detection primer set: Bst DNA polymerase: chromogenic reagent is 15:6:1:1 to prepare the falciparum malaria detection system; According to the volume ratio, LAMP amplification reaction solution: vivax malaria detection primer set: Bst DNA polymerase: chromogenic reagent 15:6:1:1 to prepare the vivax malaria detection system; According to the volume ratio, the LAMP amplification reaction solution: Plasmodium spp. detection primer set: Bst DNA polymerase: chromogenic reagent is 15:6:1:1 to prepare the Pseudomonas spp. detection system; According to the volume ratio, LAMP amplification reaction solution: Plasmodium ovale detection primer set: Bst DNA polymerase: chromogenic reagent 15:6:1:1 to prepare a detection system for Plasmodium ovale; (2.2) Place the detection system of falciparum malaria, vivax malaria detection system, triscal malaria detection system and ovale malaria detection system in four PCR tubes respectively, and add the DNA of the sample to be tested to each PCR tube, 60 React at ~65°C for 45-90 minutes; observe the color of the liquid in each PCR tube, green fluorescence is positive, and orange is negative. 6. The detection method of Plasmodium according to claim 5, wherein the process of extracting the DNA of the sample to be tested is: taking 1-5 μl of peripheral blood or 1-5 μl of filter paper dried blood and placing it in a centrifuge tube, Add 10 μl of 30% H ₂ O ₂ and 50 μl of 0.1 mol/L NaOH, lyse at 95°C for 20 min, cool to room temperature, add 50 μl of 1 mol/L Tris-HCl pH 8.0, mix well to obtain the DNA of the sample. 7. the detection method of malaria parasite according to claim 5, is characterized in that, it also comprises setting blank control group and negative control group, and step (2) is specifically, falciparum malaria detection system, vivax malaria detection system, three days Three groups of parallel experiments were set up for each system in the malaria detection system and the malaria ovale detection system. One of them was an experimental group, and the DNA of the sample to be tested was added to the system, and the other group was a negative control group, and a healthy group was added to the system. DNA from human blood, the last group is a blank control group, deionized water is added to the system; the reaction is performed at 60-65°C for 45-90 minutes; the color of the liquid in each PCR tube is observed respectively.

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