A multiplex PCR primer set and method for simultaneously detecting multiple symbiotic bacteria and viruses in bemisia tabaci

Abstract

The application discloses a multiplex PCR primer set for simultaneously detecting multiple symbiotic bacteria and viruses in Bemisia tabaci, comprising the following: a primer pair for detecting TYLCV, the nucleotide sequences of which are shown as SEQ ID NO:1 and SEQ ID NO:2; a primer pair for detecting Rickettsia , the nucleotide sequences of which are shown as SEQ ID NO:3 and SEQ ID NO:4; a primer pair for detecting Cardinium , the nucleotide sequences of which are shown as SEQ ID NO:5 and SEQ ID NO:6; and a primer pair for detecting Hamiltonella , the nucleotide sequences of which are shown as SEQ ID NO:7 and SEQ ID NO:8. The application solves the technical problems of low detection efficiency, large sample consumption, high reagent cost, inconvenient data comparison and poor compatibility of primers in a multiplex PCR system in the prior art.

Description

Technical Field

[0001] This invention relates to the field of bio-agricultural technology, specifically to a multiplex PCR primer set and method for simultaneously detecting multiple symbiotic bacteria and viruses in whiteflies. Background Technology

[0002] The whitefly is a global agricultural pest that damages its hosts by sucking plant sap, and its honeydew secretions can induce sooty mold, indirectly affecting plant photosynthesis. Furthermore, the whitefly acts as a vector for plant viruses, transmitting over 200 plant viruses and posing a serious threat to hundreds of economic crops worldwide.

[0003] Whiteflies carry a variety of symbiotic microorganisms, including primary and secondary endosymbiotics (such as...). Cardinium , Rickettsia , Hamiltonella These microorganisms, including plant viruses such as Tomato Yellow Leaf Curl Virus (TYLCV), are closely related to various biological characteristics of whiteflies, including reproductive regulation, pesticide resistance, and virus transmission capabilities. Therefore, the joint detection of these microorganisms is of significant scientific value and practical application for analyzing the genetic structure of whitefly populations, optimizing biological control strategies, and providing early warning of plant viral diseases.

[0004] Currently, the detection of symbiotic bacteria and viruses in whiteflies mainly relies on polymerase chain reaction (PCR) technology. However, existing methods mostly employ singleton PCR, meaning that each target microorganism undergoes independent PCR amplification and gel electrophoresis detection. To detect... Cardinium , Rickettsia , Hamiltonella For the four targets of TYLCV, four PCR reactions and four electrophoresis processes are required. This process is cumbersome and time-consuming, severely limiting the efficiency of processing large batches of samples. Furthermore, multiple independent detections consume large amounts of DNA template, often making it impossible to detect all targets for valuable samples such as single whiteflies, rare populations, or aged specimens with long preservation periods. In addition, the reagent consumption from multiple PCR reactions significantly increases the detection cost, and the separate electrophoresis results require manual alignment of the patterns during comprehensive analysis, which is not only inefficient but also prone to interpretation errors and recording confusion.

[0005] Although multiplex PCR detection methods for betaviruses transmitted by whiteflies have been reported in existing technologies, these methods only cover viral targets and fail to include symbiotic bacteria in the simultaneous detection system. Furthermore, for... Cardinium , Rickettsia and HamiltonellaSimultaneous detection of multiplex PCR for symbiotic bacteria has not yet been reported. Jointly detecting multiple symbiotic bacteria and TYLCV in the same reaction system requires overcoming key technical challenges in multiplex PCR, including primer dimer formation, non-specific amplification, and imbalances in amplification efficiency among different targets. Current technologies have not yet effectively solved these problems. Summary of the Invention

[0006] This application provides a method for simultaneously detecting three symbiotic bacteria in whiteflies ( Cardinium , Rickettsia , Hamiltonella This invention relates to a multiplex PCR primer set and detection method for a virus (TYLCV) to address technical problems in existing technologies, such as low detection efficiency, high sample consumption, high reagent cost, inconvenient data comparison, and poor primer compatibility in multiplex PCR systems.

[0007] To achieve the above objectives, the first aspect of this application provides the following technical solution: A set of multiplex PCR primers for simultaneous detection of multiple symbiotic bacteria and viruses in whiteflies, comprising: The primer pairs used for detecting TYLCV have the following nucleotide sequences: SEQ ID NO:1 and SEQ ID NO:2. Used for detection Rickettsia The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4; Used for detection Cardinium The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6; Used for detection Hamiltonella The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0008]

[0009] Furthermore, the primer pair for detecting TYLCV amplifies a fragment with a length of 505 bp; the detection Rickettsia The primer pair amplified a fragment with a length of 719 bp; the detection Cardinium The primer pair amplified a fragment with a length of 209 bp; the detection Hamiltonella The primer pair amplified a fragment of 420 bp.

[0010] The second aspect of this application provides the following technical solution: A method for simultaneously detecting multiple symbiotic bacteria and viruses in whiteflies includes the following steps: S1. Extract total DNA from the whitefly sample to be tested; S2. Using total DNA as a template, perform PCR amplification using the multiplex PCR primer set described in the first aspect; S3. Detect the PCR amplification products and determine whether the whitefly sample carries TYLCV based on the test results. Rickettsia , Cardinium and Hamiltonella .

[0011] The optimized final concentration of the TYLCV detection primer pair in the reaction system was 0.2 μM; detection Rickettsia The final concentration of the primer pair was 0.1 μM; detection Cardinium The final concentration of the primer pair was 0.2 μM; detection Hamiltonella The final concentration of the primer pair was 0.2 μM.

[0012] In some embodiments, the annealing temperature for the PCR amplification is 56-62°C.

[0013] A third aspect of this application provides a detection kit comprising the multiplex PCR primer set described in the first aspect.

[0014] Furthermore, this invention provides an application of multiplex PCR primer sets in the genetic analysis of whitefly populations, the formulation of biological control strategies, or disease early warning.

[0015] Working principle and beneficial effects of the present invention: 1. High throughput and high efficiency: By combining four pairs of specific primers in a single PCR reaction tube, "one tube, multiple detections" are achieved. Only one round of PCR amplification and one gel electrophoresis are required to simultaneously detect substances in whiteflies. Cardinium , Hamiltonella , Rickettsia Detection of four microorganisms including TYLCV. Compared with separate detection (requiring 4 PCRs + 4 electrophoresis), the detection throughput is increased by 4 times and the time is reduced by more than 70%.

[0016] 2. Saves samples and costs: Only a tiny amount of DNA template is needed per reaction, which is especially advantageous for precious samples. At the same time, the amount of PCR reagents (Taq enzyme, dNTPs, etc.) used is reduced to 1 / 4 of the original amount, significantly reducing detection costs.

[0017] 3. Results are intuitive and easy to interpret: The amplified fragment lengths of the four target microorganisms (209bp, 420bp, 505bp, and 719bp) showed clear spacing on the gel, and the electrophoretic bands were clearly distinguishable without overlapping interference (see [link to original text]). Figure 2 This makes result interpretation simple, intuitive, and reliable, avoiding interpretation errors or record confusion that may occur when testing separately.

[0018] 4. High specificity and high sensitivity: The primer set screened in this application maintains extremely high specificity in a multiplex PCR system, accurately distinguishing target microorganisms from non-target templates. Through system optimization, its detection sensitivity can reach a level comparable to that of single PCR, avoiding false negatives.

[0019] 5. Provides a powerful tool for in-depth research: This method provides an ideal technical means for large-scale, rapid screening of the carriage rate, co-infection patterns, and association with the biological phenotype of these four important microorganisms in whitefly populations. Attached Figure Description

[0020] Figure 1 A gel electrophoresis image of a single PCR amplification product (TYLCV) ， 505 bp; Rickettsia, 719 bp; Cardinium, 209 bp; Hamiltonella, 420 bp); Figure 2 This is a gel electrophoresis image (TYLCV) of the amplification products of the multiplex PCR method of this invention. ， 505 bp; Rickettsia, 719 bp; Cardinium, 209 bp; Hamiltonella, 420 bp). Detailed Implementation

[0021] The following detailed description illustrates the specific implementation method: Example 1: Single PCR verification of primer specificity Total DNA extraction from whiteflies: A single whitefly was placed in a 1.5 ml centrifuge tube, lysis buffer and proteinase K were added, and the mixture was digested in a 56 °C water bath until the tissue was completely lysed. Then, binding, washing and elution steps were performed sequentially to obtain approximately 50 μL of total DNA solution, which was stored at -20 °C for later use.

[0022] TYLCV positive, respectively Rickettsia Positive, Cardinium Positive, Hamiltonella Using DNA from whiteflies that were positive for the virus and negative for all four targets as templates, a standard PCR system was used, employing TYLCV-F / R, Rickettsia -F / R、 Cardinium -F / R、 Hamiltonella Four primer pairs (-F / R) were used for individual amplification, and the PCR products were detected by agarose gel electrophoresis. The primer set is shown in the table below:

[0023] The results show that: (refer to) Figure 1The primer pair amplified specific bands only in the positive templates corresponding to their respective targets, and no amplification bands were observed in other templates or negative controls. The amplified fragment sizes were approximately TYLCV 505 bp and... Rickettsia Approximately 719 bp Cardinium Approximately 209 bp Hamiltonella The length is approximately 420 bp, indicating that all four primer pairs designed in this invention have good specificity.

[0024] Example 2: Optimization of Multiplex PCR Reaction System and Annealing Temperature Using mixed positive DNA (containing all four targets) as a template, prepare 25 μL or 50 μL multiplex PCR reaction systems containing four pairs of specific primers, dNTPs, and Mg. 2+ Taq DNA polymerase and buffer were used. An annealing temperature gradient of 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, and 62℃ was set for amplification. The amplified products were detected by agarose gel electrophoresis.

[0025] The results showed that at an annealing temperature of 58-59℃, clear specific bands were obtained for all four targets, with no non-specific amplification products or primer dimers. Non-specific bands appeared when the temperature was too low, and the amplification efficiency of some targets decreased when the temperature was too high.

[0026] Figure 2 The four clearly separated target bands correspond to Cardinium (209 bp, 0.2 μM) Hamiltonella (420 bp, 0.2 μM), TYLCV (505 bp, 0.2 μM) and Rickettsia (719 bp, 0.1 μM).

[0027] Example 3: Optimization of multiplex PCR primer concentrations Based on the reaction system determined in Example 2, the final concentrations of the four primer pairs were optimized using gradient methods. The concentrations of each primer pair were adjusted according to the differences in amplification efficiency to ensure a balanced amplification product yield for the four targets. Using the uniformity of grayscale values ​​of each band as an evaluation index, orthogonal experiments were conducted to determine the optimal concentration ratio of each primer pair.

[0028] The results showed that when the final concentration of the TYLCV primer pair was 0.2 μM, Rickettsia The final concentration of the primer pair was 0.1 μM. Cardinium The final concentration of the primer pair was 0.2 μM. Hamiltonella When the primer pair concentration was 0.2 μM, the band brightness of the amplification products for the four targets was most uniform. This concentration ratio effectively overcomes the problem of uneven amplification efficiency of different targets in multiplex PCR.

[0029] Example 4: Sensitivity Validation of Multiplex PCR Based on the optimal reaction system and annealing temperature (58-59℃) determined in Example 2, the mixed positive DNA (containing all four targets) was serially diluted to obtain a series of template concentrations: 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL. Multiplex PCR amplification was performed using each of these DNA concentrations as templates, with three replicates for each concentration. The amplification products were detected by agarose gel electrophoresis.

[0030] Agarose gel electrophoresis results showed that this method is effective for TYLCV (505 bp). Cardinium (209 bp) and Hamiltonella The detection sensitivity of all three targets (420 bp) reached 10 pg / μL DNA template. At this concentration, identifiable specific amplification bands (weak in brightness but clearly distinguishable) were obtained for all three targets. Rickettsia (The longest amplified fragment, 719 bp) was undetectable at this concentration. The difference in detection sensitivity between different targets is mainly related to the length of their amplified fragments—the longer the fragment, the lower the amplification efficiency. In addition, it may also be related to factors such as the binding efficiency of each primer pair and the relative copy number of the target in the mixed template.

[0031] When the DNA template concentration dropped to 1 pg / μL or below, none of the four targets could be detected, indicating that this concentration exceeded the detection limit of this method. No amplification bands were observed in the negative controls, ruling out the possibility of reagent contamination.

[0032] Example 5: Application of detection in field samples of whiteflies Whitefly field populations were collected from different regions. 20-30 single adult whiteflies were randomly selected from each population, and DNA was extracted from each. The DNA was then detected using the multiplex PCR method established in this invention. At the same time, a single PCR method was used for parallel verification.

[0033] The results showed that the method of the present invention achieved effective amplification in all samples, and the specific bands of the four targets were clearly distinguishable. Different samples showed diverse infection patterns (quadruple positive, triple positive, double positive, single positive, and all negative). The target detection data were compared with the single PCR results, and the concordance rate was 100%.

[0034] Taking the testing of 96 samples as an example, the method of this invention requires only 96 PCR tubes and one electrophoresis detection, while the single PCR method requires 384 PCR tubes and four electrophoresis detections. The method of this invention saves approximately 75% in terms of reagent consumption, operation time, and labor costs, significantly improving detection efficiency. The detection results were verified by sequencing, showing that the sequences of each target amplification product were consistent with the target sequence, further confirming the accuracy of this invention.

[0035] The above description is merely a preferred embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the inventive concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A multiplex PCR primer set for simultaneous detection of multiple symbiotic bacteria and viruses in Bemisia tabaci, characterized in that, include: The primer pairs used for detecting TYLCV have nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2; a primer pair for detecting Rickettsia the nucleotide sequences of which are shown as SEQ ID NO: 3 and SEQ ID NO: 4; Used for detection Cardinium The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6; Used for detection Hamiltonella The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:

8.

2. The multiplex PCR primer set according to claim 1, characterized in that, The primer pair for detecting TYLCV amplifies a fragment of 505 bp in length; the detection Rickettsia The primer pair amplified a fragment with a length of 719 bp; the detection Cardinium The primer pair amplified a fragment with a length of 209 bp; the detection Hamiltonella The primer pair amplified a fragment of 420 bp.

3. A method for simultaneously detecting multiple symbiotic bacteria and viruses in whiteflies, characterized in that, Includes the following steps: S1. Extract total DNA from the whitefly sample to be tested; S2. Using total DNA as a template, perform PCR amplification using the multiplex PCR primer set as described in claim 1 or 2; S3. Detect the PCR amplification products and determine whether the whitefly sample carries TYLCV based on the test results. Rickettsia , Cardinium and Hamiltonella .

4. The detection method according to claim 3, characterized in that, The final concentration of the TYLCV primer pair in the reaction system was 0.1-0.3 μM; detection Rickettsia The final concentration of the primer pairs was 0.05-0.25 μM; detection Cardinium The final concentration of the primer pairs was 0.1-0.25 μM; detection Hamiltonella The final concentration of the primer pairs is 0.1-0.3 μM.

5. The detection method according to claim 4, characterized in that, The annealing temperature for the PCR amplification is 56-62℃.

6. A test kit, characterized in that, It includes the multiplex PCR primer set as described in claim 1 or 2.

7. The application of the multiplex PCR primer set according to claim 1 or 2 in the genetic analysis of whitefly populations, the formulation of biological control strategies, or disease early warning.

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