CN111534626A - A LAMP detection primer composition, detection kit and visual detection method for Pythium belligerents - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to an LAMP (loop-mediated isothermal amplification) detection primer composition, a detection kit and a visual detection method for pythium bellatus, wherein genomic DNA of a microorganism to be detected is extracted, the genomic DNA solution is taken as a reaction template, and the detection solution in the LAMP kit is added for LAMP reaction, and the LAMP reaction is carried out by the following procedures: and (3) carrying out reaction amplification at 64 ℃ for 60min, and observing the color change of an amplification product, wherein if the color is changed from purple to blue, the pythium clotocidum exists in the object to be detected, and if the color is not changed and is still purple, the pythium clotocidum does not exist in the object to be detected. Compared with the traditional detection technology depending on morphology and molecular PCR, the method has higher accuracy, sensitivity and effectiveness, and is simple to operate. The method can be used for quickly detecting the pythium closterium and provides technical support for monitoring and preventing diseases caused by the pythium closterium.

Description

A LAMP detection primer composition for Pythium bellum, detection kit and the same Visual detection method

technical field

The invention relates to the field of biotechnology, in particular to a LAMP detection primer composition, a detection kit and a visual detection method thereof for Pythium belligerents.

Background technique

As an important pathogen of oomycetes, Phytopythium vexans can infect tobacco, citrus, ramie and other important economic crops. Ramie is a perennial herb of the genus Ramie of the Urticaceae family, native to East Asia, and one of the oldest fiber crops in the world, with high economic value. The root brown rot mainly caused by Pythium bellum is a new devastating disease of ramie roots discovered in recent years. A method that can quickly and accurately identify the pathogenic bacteria in the early stage of ramie onset. At present, the identification method of Pythium bellum is mainly through the cultivation of pathogenic bacteria and observation and identification. However, due to the difficulty of isolation and cultivation of Pythium, the hyphae, sporangia and oospores of closely related species are very similar in morphology, and the diseases caused by Pythium are very similar. The symptoms are similar to other plant diseases, and it is difficult to distinguish, which makes the identification of the fungus very difficult. It can be seen that the traditional identification method requires rich knowledge and experience of pathogen morphological identification, which is not only time-consuming, but also difficult to judge the accuracy, which is not conducive to the analysis and identification of large-capacity samples, and cannot meet the needs of field production. With the development of molecular biology, a variety of detection technologies based on polymerase chain reaction (PCR) and real-time quantitative PCR have been developed and widely used, and have become the main methods for rapid, accurate and specific identification of pathogenic bacteria. means to avoid the disadvantage of uncertainty in morphological identification. However, these detection techniques often need to be carried out in laboratories with professional instruments, reagents and strict environmental conditions. The instruments and reagents are expensive and require high operation, the process is complicated, and the detection time is still long. These shortcomings make them unsuitable in Use in grassroots environments.

Loop-mediated isothermal amplification (LAMP) is a newly developed molecular detection technology that uses four specific primers and Bst DNA polymerase with strand displacement activity to achieve target detection under constant temperature conditions. Mass amplification of DNA in a short period of time. There are many ways to test the results of LAMP technology. Under high-efficiency amplification conditions, the pyrophosphate ions precipitated from the deoxynucleic acid triphosphate matrix (dNTPs) combine with the magnesium ions in the system to produce a large amount of magnesium pyrophosphate, resulting in a white appearance. Therefore, the amplified product can be monitored by turbidimeter; it can also be detected by gel electrophoresis, or the fluorescent dye calcein or hydroxynaphthol blue (HNB) can be added to the reaction system. You can observe and judge the test results. This method does not require professional instruments and complicated procedures, and has the advantages of simple operation, rapidity and efficiency, strong specificity and high sensitivity, and is a rapid detection technology suitable for grass-roots use. LAMP technology has been widely used in the rapid detection of fungi, bacteria, viruses, nematodes and other pathogens.

In view of this, the present invention uses the ITS sequence as the target sequence for detection, designs LAMP primers specific to Pythium belligerents, and establishes a rapid LAMP detection method for Pythium belligerents on this basis.

SUMMARY OF THE INVENTION

In view of the problems in the prior art that the biological detection method of Pythium belligerents requires a long period of time, the detection method has poor specificity and low sensitivity, the purpose of the present invention is to provide a LAMP detection primer composition and reagent for Pythium belligerents Box and its visual detection method.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

A LAMP detection primer composition for Pythium bellumensis, the primer composition comprises: forward outer primer F3 as shown in SEQ ID NO:1, reverse outer primer B3 as shown in SEQ ID NO:2 , the forward inner primer FI shown in SEQ ID NO:3, and the reverse inner primer BIP shown in SEQ ID NO:4.

The present invention also provides a LAMP detection kit for Pythium belligerents, where the LAMP detection reagent includes the above-mentioned LAMP detection primer composition.

Preferably, the LAMP detection kit further includes one or more of Bst DNA polymerase, reaction buffer, MgSO ₄ , dNTP, betaine, and dye indicator.

Preferably, the LAMP detection reagent also includes water.

The present invention also provides a LAMP detection method of Pythium belligerents, which uses the above-mentioned LAMP detection primers to amplify the DNA of Pythium belligerents.

The reaction system of the LAMP detection method is as follows: 1.6 μmol/L each of FIP and BIP, 0.2 μmol/L each of F3 and B3, 8UBst DNA polymerase, 2.5 μL 10× reaction buffer, 6 mmol/L MgSO ₄ , 1.4 mmol/L dNTPs , 0.8mol/L betaine, 180μmol/L staining indicator, 2μL DNA template, sterilized water to make up to 25μL.

The reaction temperature of LAMP was 64°C, and the reaction time was 60 min.

When the dyeing indicator is hydroxynaphthol blue, the judgment method of the LAMP detection method is as follows: the color of the reaction solution is purple, indicating that the result is negative, and the sample to be tested does not contain Pythium bellum; the color of the reaction solution is blue, indicating that the result is Positive, the sample to be tested contains Pythium bell.

Compared with the prior art, the present invention has the following advantages and positive effects:

1. Easy to operate: The LAMP method for detecting Pythium bellum provided by the present invention overcomes the problems of long period, time-consuming, laborious, cumbersome, poor specificity and PCR detection technology of the biological detection method of Pythium bellum in the prior art. Requires thermal cycling instrumentation and cannot quickly detect the problem of Pythium in bells. Under the isothermal condition of 64°C, the detection method of the invention can accurately, quickly and efficiently detect Pythium belligerents within 60 minutes, has simple requirements on the experimental site, does not require other complicated instruments, and can better satisfy the requirements of Pythium bellum It is suitable for promotion and use in grass-roots departments.

2. High accuracy: Because the traditional detection method of Pythium bell is mainly through the cultivation of pathogenic bacteria and observation and identification, but due to the difficulty of isolation and cultivation of Pythium, the hyphae, sporangia and oospores of closely related species are very similar in morphology. , and the disease symptoms caused by it are similar to other plant diseases, which are difficult to distinguish, which makes the identification of the fungus very difficult. In the present invention, according to the genome sequence of Pythium belligerents, Megalign software is used to compare it with other Pythium spp. genomes, and genes with greater differences between Pythium belligerents and other strains are selected as target genes to design specific LAMP primers. The LAMP reaction specifically recognizes 6 independent regions on the target sequence through 4 primers (FIP, BIP, F3, B3), and its specificity is relatively high. In addition, in the present invention, a dye indicator is added in advance to the LAMP reaction system, so that the LAMP detection result can be judged by visual observation, and the possibility of contamination by opening the cap is avoided.

3. High sensitivity: The LAMP detection method of Pythium bellum established by the present invention has very high sensitivity. The amplification product can be detected when the DNA concentration is between 10ng/μL and 1pg/μL, and the color of the system changes to different degrees of blue. color, indicating that the detection method is sufficient for accurate and rapid detection of Pythium belligerents at low DNA concentrations.

Description of drawings

Fig. 1 Morphology of Pythium colony on the bell;

Figure 2 Specific detection of Pythium bellum LAMP primers; among them, (a): 2% agarose gel electrophoresis detection results; (b): hydroxynaphthol blue (HNB) visual color detection results, the results show that only clock A blue positive reaction can be observed for Pythium vulgaris, while a purple negative reaction is observed for other pathogens.

Fig. 3 Comparison of the sensitivity of conventional PCR and LAMP detection methods of Pythium bellum; wherein, (a): 2% agarose gel electrophoresis detection result of conventional PCR amplification products; (b): 2% agarose of LAMP amplification products Glycogel electrophoresis detection results; (c): hydroxynaphthol blue (HNB) visual color detection results of LAMP amplification products; DNA concentrations ranged from 10ng/μL to 1fg/μL, the results showed that the conventional PCR of Pythium belligerents was effective. The minimum detection limit is 100pg/μL, and the minimum detection limit of the LAMP detection system of Pythium bellum is 1pg/μL;

Fig. 4 Ramie diseased roots infected with different Pythium;

Figure 5. LAMP detection of ramie roots infected with different Pythium species; among them, (a): 2% agarose gel electrophoresis detection results; (b): hydroxynaphthol blue (HNB) visual color detection results, the results show clock The DNA system of the ramie root infected by Pythium vulgaris had a band, and the color development result of the LAMP detection system was a blue positive reaction; the other ramie root DNA systems infected by Pythium had no band, and the color development result of the LAMP detection system was as follows: Purple negative reaction.

Detailed ways

The invention discloses a LAMP detection primer composition, a detection kit and a visual detection method for Pythium belligerents, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The LAMP detection primers, detection reagents, and reagents or instruments used in the detection method for Pythium belligerents provided by the present invention can be purchased from the market. See Figure 1 for the colony morphology of the isolated Pythium bellum according to the present invention.

Below in conjunction with embodiment, the present invention is further elaborated:

Embodiment 1 utilizes the LAMP method to detect the Pythium of the clock

LAMP primer composition for detecting Pythium bellum: forward outer primer F3 as shown in SEQ ID NO: 1, reverse outer primer B3 as shown in SEQ ID NO: 2, forward inner primer FI as shown in SEQ ID NO: 1 : 3, the reverse inner primer BIP is shown in SEQ ID NO: 4.

The concentrations of each reagent in the LAMP kit for detection of Pythium bellum are: 1.6 μmol/L each of FIP and BIP, 0.2 μmol/L each of F3 and B3, 8 U Bst DNA polymerase, 2.5 μL 10× reaction buffer, 6 mmol/L MgSO ₄ , 1.4mmol/LdNTPs, 0.8mol/L betaine, 180μmol/L staining indicator, 2μL DNA template, sterile water to make up to 25μL.

LAMP detection method: extract the DNA of the microorganism to be detected, take 2 μL of DNA template, add 23 μL of the detection solution in the LAMP kit for LAMP detection, and react at 64°C for 60 min. Then observe the color change of the product, the color of the reaction solution is purple, indicating that there is no Pythium bell in the sample to be tested, and the color of the reaction solution is blue, indicating that the sample to be tested contains Pythium bell.

In order to verify the specificity of the LAMP detection method, 2 strains of Pythium bellum, 13 strains of Pythium relatives and 5 other pathogenic fungi were used as test materials. The species, host, source and quantity of the tested strains are shown in Table 1. The LAMP test results showed that Pythium bellum could be observed as a positive reaction in blue, while other pathogens showed a negative reaction in purple (Figure 2).

Table 1 Test strains and sources for LAMP detection

Embodiment 2 Sensitivity detection of Pythium bellum LAMP reaction

In order to determine the sensitivity of the LAMP detection method, the extracted DNA of Pythium belligerents was measured with a NanoDrop 2000 ultra-micro spectrophotometer and then diluted 10 times to the range of 10 ng/μL～1fg/μL. The LAMP amplification reaction was performed as a template, and a negative control was set. After the reaction, the color change was observed according to the HNB visual detection method, and the minimum detection limit of the LAMP reaction was judged, which was verified by 2% agarose gel electrophoresis. The results showed that the amplification products could be detected by conventional PCR detection DNA concentration between 10ng/μL and 100pg/μL, but when diluted to 100 pg/μL, the brightness of the electrophoresis band detected by the gel was extremely weak, and the dilution was continued to 100 pg/μL. The amplification product could not be detected at 10 pg/μL, which proved that the minimum detection limit of conventional PCR for Pythium belligerents was 100 pg/μL [Fig. 3(a)]. When verifying the sensitivity of the LAMP detection method, it was found that the amplification product could be detected at the DNA concentration between 10ng/μL and 1pg/μL, and the color of the system changed to different degrees of blue. Positive result; when the concentration is diluted to 100fg/μL, the color of the system no longer changes, it is still negative in purple, and there is no electrophoresis band, so 1pg/μL is the minimum detection limit of the LAMP detection system of Pythium bellum chinensis [Figure 3( b), (c)]. The above results prove that the sensitivity of the Pythium bellum LAMP detection system established by the present invention is at least 100 times that of conventional PCR.

Example 3 Strain back connection and ramie tissue LAMP detection

Asexual reproduction of ramie: Cut off the stalks with terminal buds from the vigorous and tall plants in the soil-cultivated ramie, select the ramie stalks with moderate thickness and no bending, and insert them into a clean 100mL conical flask, Add an appropriate amount of water, put it in a greenhouse at 25°C for cultivation, and select healthy plants with similar growth conditions for back-connection experiments after growing fibrous roots.

Strain back connection: 15 kinds of Pythium in the test strains in Table 1 were activated and cultivated on PDA medium 3 to 4 days later, use a hole puncher to punch the fungus cake, and connect them back to the ramie grown by hydroponic culture respectively. On the white fibrous roots, the mycelium was close to the fibrous roots, and each ramie root was connected to 4 bacterial cakes of the same Pythium, and cultured at 25 °C in the greenhouse for 7 to 10 days. .

The LAMP reaction system was constructed with the diseased plant tissue DNA as the template, and the amplified products were detected by two methods: HNB visual color detection and 2% agarose gel electrophoresis detection, to further verify the accuracy of the LAMP reaction system of Pythium belligerents. .

After 15 days of culture, hydroponic ramie grew long white fibrous roots, and the activated test strains were pasted on the densely growing fibrous roots. After culturing at 25 °C for 10 days, the ramie fibrous roots infected by different strains showed In different disease states, the diseased roots changed from normal white to different degrees of brownish yellow, and the fibrous roots of the control group were still white (Figure 4).

Cut the ramie roots around the bacterial cake into pieces and put them into centrifuge tubes, divide into 2 tubes, each tube weighs about 0.2g of fibrous roots, extract its DNA, and use it as a template to construct a LAMP reaction system to test the accuracy of the reaction. . After the reaction, by observing the color of the system, it can be seen that the LAMP system using the ramie root DNA infected with the two species of Pythium bellum as the template is blue positive, and the color of the system containing the rest of the ramie root DNA is purple negative (Fig. 5(b) ) ]. The electrophoresis detection also showed the same result. Except for Pythium bellum, the other Pythium-infected ramie root DNA systems showed no bands [Fig. 5(a)], indicating the LAMP amplification reaction using this primer. The method can accurately detect Pythium bell in the roots of ramie, and can be used for the detection of Pythium bell in the ramie root brown rot diseased plants in the field.

Comparative Examples 1-8

The primer sequences of Comparative Examples 1-8 are as follows:

Table 2 LAMP primers of Pythium belligerents of Comparative Examples 1-8

Test Example 1 Specificity Verification of Different Primer Sets

The 20 fungal ITS regions listed in Table 1 were amplified by PCR, and 9 groups of primers were designed using the online primer design software PrimerExplorerV5 for the Pythium bellum ITS sequence screened after sequencing and comparison (see Example 1, Comparative example 1-8 primer set sequence), each set includes a pair of outer primers F3, B3 and a pair of inner primers FIP, BIP, namely 4 LAMP-specific primers.

Set up a LAMP reaction system, including: outer primers F3 and B3 each 0.2μmol/L, inner primers FIP and BIP each 1.6μmol/L, 2.5μL 10×Isothermal Amplification Buffer [containing 20mmol/L Tris-HCL (PH8.8), _10mmol /L( _NH4 ) _2SO4 , 50mmol/L KCL, 0.1%Tween-20], 1.4mmol/L dNTPs, 6mmol/L _MgSO4 , 0.8mol/L betaine, 8U Bst DNA polymerase, 180μmol/ L Hydroxynaphthol blue (HNB), 2 μL DNA template, make up to 25 μL with sterile water. Hydroxynaphthol blue is used as indicator in the system, purple is negative reaction, blue is positive reaction.

The above system was constructed with Pythium bellum DNA as a template, and the primers were verified in a water bath at 64 °C for 60 min; after taking out, observe the color change of the system, if the color changes from purple to blue, it proves that the primers were successfully amplified. Pythium bellum DNA, which can be further optimized; if the color does not change, the primer is not usable. It can also be verified by 2.0% agarose gel electrophoresis. If a ladder band appears, the primer is available, otherwise the primer is not available.

The results showed that the primer set of Example 1 had the highest specificity, electrophoresis showed that no bands were produced in the other pathogens except Pythium bell, and the LAMP results showed that only Pythium bell could observe a blue positive reaction, while Other pathogenic bacteria showed negative reaction in purple (Figure 2).

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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Claims (8)

1. An LAMP detection primer composition for pythium bellatus, which is characterized in that the sequence of the primer is as follows:

forward outer primer F3: the sequence is shown as SEQ ID NO: 1 is shown in the specification;

reverse outer primer B3: the sequence is shown as SEQ ID NO: 2 is shown in the specification;

forward inner primer FIP: the sequence is shown as SEQ ID NO: 3 is shown in the specification;

reverse inner primer BIP: the sequence is shown as SEQ ID NO: 4, respectively.

2. An LAMP detection kit for Pythium closterium, which is characterized in that the LAMP detection reagent comprises the LAMP detection primer composition of claim 1.

3. The LAMP detection kit according to claim 2, wherein the LAMP detection reagent further comprises BstDNA polymerase, reaction buffer, MgSO₄One or more of dNTP, betaine and a staining indicator.

4. The LAMP detection kit according to claim 3, characterized in that the LAMP detection reagent further comprises water.

5. An LAMP detection method of Pythium closterium, characterized in that DNA of Pythium closterium is amplified by using the LAMP detection primer composition according to any one of claims 1 to 4.

6. The LAMP detection method according to claim 5, wherein the LAMP detection method comprises the reaction system of 1.6. mu. mol/L for FIP and BIP, 0.2. mu. mol/L for F3 and B3, 8U BstDNA polymerase, 2.5. mu.L 10 × reaction buffer, 6mmol/L MgSO₄1.4mmol/L dNTPs,0.8mol/L betaine, 180 mu mol/L staining indicator, 2 mu L DNA template and sterile water to 25 mu L.

7. The LAMP detection method according to claim 6, wherein the LAMP has a reaction temperature of 64 ℃ and a reaction time of 60 min.

8. The LAMP detection method according to claim 7, wherein the staining indicator is hydroxynaphthol blue, and the LAMP detection method is characterized in that:

the color of the reaction solution is purple, the result is negative, and the sample to be detected does not contain pythium clotocaly;

the color of the reaction solution is blue, the result is positive, and the sample to be detected contains pythium closterium.

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