Establishment of a Rapid Detection Method for Yeast-like Symbionts in Brown Planthopper Based on Droplet Digital PCR Technology
Abstract
:1. Introduction
2. Results
2.1. Results of Primer Screening for PCR and RT-qPCR Amplification
2.2. Optimization of ddPCR System
2.2.1. Optimization of Primer Concentration
2.2.2. Optimization of Probe Concentration
2.3. ddPCR Sensitivity Test
2.4. ddPCR Specificity Test
2.5. ddPCR Repeatability Test
3. Discussion
4. Materials and Methods
4.1. Collection and Culture of Insects
4.2. Specific Experiment
4.2.1. Principal Reagent
4.2.2. Main Instruments and Materials
4.3. Genome Extraction of Symbiotic Fungi
4.4. Design and Screening of Primer and Probe
4.4.1. PCR Amplification and Sequencing Alignment
4.4.2. SYBR Green Dye Real-Time Quantitative PCR
4.4.3. Optimization of Annealing Temperature Using Taq Man Probe Real-Time Quantitative PCR
4.5. Plasmid Sample Preparation
4.6. ddPCR System Optimization
4.7. Sensitivity Test
4.8. Specific Test
4.9. Data Statistics and Analysis
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Symbionts | Primers | Sequences (5′ → 3′) | Amplification Product Size (bp) |
---|---|---|---|
Ascomycetes symbionts | AsF | GTCGTAGTCTTAACCATAA | 145 |
AsR | CTTCCGTCAATTTCTTTAAG | ||
AsP | TCAGCCTTGCGACCATA | ||
Pichia guilliermondii | PgF | CCTCTCAATGTATTAGGTTTA | 101 |
PgR | TGAGGTCAAACTTGTTTG | ||
PgP | CCAACAATACCAGAAATATCCCGCC |
Symbionts | Primer Concentration (nM) | |||||
---|---|---|---|---|---|---|
600 | 700 | 800 | 900 | 1000 | ||
Ascomycetes symbionts | Copy number copies/μL | 903.00 | 962.00 | 965.00 | 963.00 | 965.00 |
Pichia guilliermondii | Copy number copies/μL | 73.00 | 80.00 | 79.60 | 134.00 | 76.00 |
Symbionts | Probe Concentration (nM) | |||||
---|---|---|---|---|---|---|
62.5 | 125 | 250 | 375 | 500 | ||
Ascomycetes symbionts | Copy number copies/μL | 81.10 | 89.00 | 100.00 | 92.20 | 97.00 |
Pichia guilliermondii | Copy number copies/μL | 79.20 | 78.90 | 74.90 | 68.80 | 77.50 |
Dilution Ratio | ddPCR/(copies·μL−1) | ||
---|---|---|---|
Result 1 | Result 2 | Result 3 | |
10−5 | 401.00 | 468.00 | 630.00 |
10−6 | 42.90 | 46.40 | 54.00 |
10−7 | 9.30 | 10.30 | 13.60 |
10−8 | 1.80 | 1.70 | 1.50 |
10−9 | 1.30 | 1.40 | No call |
Dilution Ratio | ddPCR/(copies·μL−1) | ||
---|---|---|---|
Result 1 | Result 2 | Result 3 | |
10−5 | 475.00 | 464.00 | 520.00 |
10−6 | 40.90 | 84.00 | 85.00 |
10−7 | 7.70 | 9.10 | 9.70 |
10−8 | 4.60 | 5.90 | 6.90 |
10−9 | 1.70 | No call | 1.20 |
Sample | Ascomycetes Symbionts | Arsenophonus Symbionts | Acinetobacter soli | Pichia guilliermondii | ddH2O |
---|---|---|---|---|---|
Copy number copies/μL | 20.50 | 2.60 | 1.70 | 3.60 | 0.80 |
22.10 | 1.40 | 1.40 | 5.00 | 0.65 | |
23.10 | 1.80 | 1.80 | 5.30 | No call |
Sample | Ascomycetes Symbionts | Arsenophonus Symbionts | Acinetobacter soli | Pichia guilliermondii | ddH2O |
---|---|---|---|---|---|
Copy number copies/μL | 2.20 | 2.40 | 0.66 | 64.20 | 0.56 |
1.50 | No call | 0.90 | 63.00 | 0.80 | |
2.00 | 1.70 | 1.30 | 71.00 | 0.90 |
Symbionts | N | Average Value (copies/μL) | Standard Deviation (copies/μL) | Relative Standard Deviation (%) | Results of One-Way ANOVA |
---|---|---|---|---|---|
Ascomycetes symbionts | 1 | 84.00 | 2.65 | 3.15 | F = 1.915 p = 0.227 |
2 | 80.00 | 2.00 | 2.50 | ||
3 | 76.33 | 2.52 | 3.30 | ||
Pichia guilliermondii | 1 | 35.67 | 1.53 | 4.28 | F = 1.126 p = 0.385 |
2 | 35.50 | 0.53 | 1.49 | ||
3 | 36.62 | 0.54 | 1.47 |
Symbiotic Bacteria | Primer | Sequence (5′-3′) | Length of Amplified Product (bp) |
---|---|---|---|
Arsenophonus symbionts | ArF | GGGAATATTGCACAATGG | 125 |
ArR | CGTCAATTGCTAAGGTTA | ||
ArP | AACCTTAACACCTTCCTCACGACT | ||
Acinetobacter soli | AcF | GCCAATTAAGTCAAATGTG | 102 |
AcR | GCTACACCTGGAATTCTA | ||
AcP | CCACACTCTAGCCAACCAGTATCG |
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Zhang, J.; Lai, C.; Shentu, X.; Hao, P.; Pang, K.; Yu, X. Establishment of a Rapid Detection Method for Yeast-like Symbionts in Brown Planthopper Based on Droplet Digital PCR Technology. Int. J. Mol. Sci. 2023, 24, 11071. https://doi.org/10.3390/ijms241311071
Zhang J, Lai C, Shentu X, Hao P, Pang K, Yu X. Establishment of a Rapid Detection Method for Yeast-like Symbionts in Brown Planthopper Based on Droplet Digital PCR Technology. International Journal of Molecular Sciences. 2023; 24(13):11071. https://doi.org/10.3390/ijms241311071
Chicago/Turabian StyleZhang, Jun, Chengling Lai, Xuping Shentu, Peiying Hao, Kun Pang, and Xiaoping Yu. 2023. "Establishment of a Rapid Detection Method for Yeast-like Symbionts in Brown Planthopper Based on Droplet Digital PCR Technology" International Journal of Molecular Sciences 24, no. 13: 11071. https://doi.org/10.3390/ijms241311071