Streamlining the Identification of the Orange Spiny Whitefly, Aleurocanthus spiniferus (Hemiptera: Aleyrodidae), with Real-Time PCR Probe Technology
Abstract
:1. Introduction
2. Materials and Methods
2.1. Insect Sampling and Identification
2.2. DNA Extraction
2.3. Design of A. spiniferus Primers and Probes
2.4. Real-Time PCR Optimization
2.5. Performance Characteristics
- -
- Analytical specificity: comparing both the tested targets (inclusivity) and non-targets (exclusivity) in the same amplification reactions with a real-time PCR, tested using normalized genomic DNA from target and non-target organisms at a final concentration of 10 ng/µL.
- -
- Analytical sensitivity (limit of detection—LoD) was assessed using a 10-fold 1:5 serial dilution in triplicate of genomic DNA extracts from single adults. The evaluation range was between 5 ng/µL and 5.12 fg/µL. All dilution measurements were performed with QIAxpert (Qiagen Hilden, Germany). Mean Cq values and their standard deviations (SDs) were calculated for the target species.
- -
- For the repeatability test, the diagnostic protocols were tested on 8 adult samples of A. spiniferus in triplicate. The reproducibility test was carried out on the same samples by two different operators on different days as in [31,38]. DNA extracts were normalized to a concentration of 0.04 ng/µL (corresponding to the third serial dilution used in the analytical sensitivity test).
3. Results
3.1. DNA Extraction
3.2. TaqMan Probe Protocol Real-Time PCR Optimization
3.3. Performance Characteristics
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | Code | Coordinates | Host Plant | Stage | Supplier | Cq ± SD |
---|---|---|---|---|---|---|
Aleurocanthus spiniferus | A.S. AA 142 | 40°52′ N; 14°29′ E | Vitis vinifera | A | IPSP | 20.23 ± 0.12 |
A.S. AA 143 | IPSP | 20.34 ± 0.28 | ||||
A.S. AA 144 | 40°52′ N;14°30′ E | Prunus sp. | N1 | IPSP | 23.02 ± 0.11 | |
A.S. AA 145 | IPSP | 22.31 ± 0.27 | ||||
A.S. AA 146 | 40°50′ N; 14°31′ E | Citrus reticulata | N1 | IPSP | 25.65 ± 0.33 | |
A.S. AA 149 | 40°52′ N; 14°29′ E | Citrus × paradisi | N2 | IPSP | 20.14 ± 0.37 | |
A.S. AA 154 | 40°40′ N; 14°45′ E | Vitis sp. | A | IPSP | 20.21 ± 0.21 | |
A.S. AA 155 | N3 | IPSP | 23.67 ± 0.34 | |||
A.S. AA 156 | Citrus sp. | IPSP | 22.65 ± 0.21 | |||
A.S. AA 158 | Edera helix | IPSP | 21.23 ± 0.26 | |||
A.S. AA 159 | A | IPSP | 25.98 ± 0.18 | |||
A.S. AA 160 | 40°44′ N; 14°38′ E | Rosa sp. | P | IPSP | 24.23 ± 0.15 | |
A.S. AA 161 | A | IPSP | 21.56 ± 0.07 | |||
A.S. AA 162 | A | IPSP | 22.54 ± 0.08 | |||
A.S. AA 164 | 40°52′ N; 14°29′ E | V. vinifera | A | IPSP | 23.65 ± 0.13 | |
A.S. AA 166 | 40°52′ N; 14°30′ E | Prunus sp. | P | IPSP | 23.45 ± 0.09 | |
A.S. AA 168 | Citrus × limon | IPSP | 23.76 ± 0.11 | |||
A.S. AA 170 | 40°40′ N; 14°45′ E | Vitis sp. | N2 | IPSP | 22.87 ± 0.14 | |
A.S. AA 171 | Citrus sp. | IPSP | 22.43 ± 0.16 | |||
A.S. AA 504 | 40°42′ N; 14°42′ E | Citrus sp. | N3 | IPSP | 21.56 ± 0.18 | |
A.S. AA 509 | 40°50′ N; 14°31′ E | Citrus sp. | N2 | IPSP | 25.76 ± 0.13 | |
A.S. AA 510 | 45°04′ N; 11°47′ E | Citrus sp. | P | IPSP | 21.23 ± 0.15 | |
A.S. AA 511 | LC | IPSP | 25,56 ± 0.43 | |||
MR4110 | 43°50′ N; 11°09′ E | Rosa sp. | LC | PPS-T | 26.71 ± 0.38 | |
MR4111 | 43°51′ N; 11°05′ E | Citrus sp. | LC | PPS-T | 25.21 ± 0.37 | |
MR4112 | 44°03′ N; 10°03′ E | Citrus sp. | LC | PPS-T | 24.21 ± 0.52 | |
MR4113 | 43°46′ N; 11°17′ E | Prunus sp. | LC | PPS-T | 25.34 ± 0.47 | |
MR4114 | 42°24′ N; 11°12′ E | Citrus sp. | LC | PPS-T | 24.46 ± 0.46 | |
MR4115 | 43°13′ N; 10°35′ E | V. vinifera | LC | PPS-T | 25.76± 0.54 | |
A.S. 1_a | 37°13′ N; 14°32′ E | Citrus sp. | A | UC | 21.34 ± 0.24 | |
A.S. 1_b | Citrus sp. | N3 | UC | 23.75 ± 0.21 | ||
A.S. 2 | 40°35′ N; 17°05′ E | Citrus sp. | N2 | UC | 23.91 ± 0.45 | |
A.S. 3 | 40°35′ N; 16°58′ E | Citrus sp. | N2 | UC | 22.41 ± 0.23 | |
A.S. 4 | 41°25′ N; 12°57′ E | Citrus sp. | N3 | UC | 23.87 ± 0.14 | |
Aleurocanthus camelliae | LabDB_3080 | 43°56′ N; 10°53′ E | Camellia sasanqua | A | PPS-T | N/A |
MR 001683 | A | PPS-T | N/A | |||
MR 001684 | 43°54′ N; 10°58′ E | A | PPS-T | N/A | ||
Planococcus citri | MR 001685 | 43°33′ N; 10°19′ E | Citrus sp. | A | PPS-T | N/A |
MR 001686 | 43°09′ N; 10°36′ E | A | PPS-T | N/A | ||
MR 001687 | 42°26′ N; 11°13′ E | Citrus sp. | A | PPS-T | N/A | |
Dialeurodes citri | LabDB_3086 | 43°43′ N; 10°21′ E | Citrus sp. | A | PPS-T | N/A |
Planococcus ficus | LabDB_3126 | 43°51′ N; 10°15′ E | Vitis sp. | A | PPS-T | N/A |
Ricania speculum | In017In015C2 | 44°16′ N; 9°27′ E | Laurus nobilis | A | PPS-T | N/A |
Saissetia oleae | In062In049C2 | 43°46′ N; 11°13′ E | Olea europaea | A | PPS-T | N/A |
In066In052C2 | A | PPS-T | N/A |
Primer/Probe Name | Length (Bases) | Sequence 5′–3′ | Nucleotide Position | Product Size (bp) | Reference Sequence |
---|---|---|---|---|---|
Aspin_10F | 19 | GCCGTTATTCTGATTATGG | 10–29 | 112 | MN662925 |
Aspin_121R | 22 | GACTCCATCACTAAATAGTAAA | 121–99 | ||
Aspin_81P | 26 | FAM—AACTTAACAGCCTGCCTATAGATGAA—BHQ1 | 81–55 |
Developmental Stage | DNA Concentration (ng/µL) ± SD | A260/280 Ratio | Cq (18S) | Cq qPCR Probe (Aleu. spiniferus) |
---|---|---|---|---|
Adults | 36.1 ± 4.53 | 1.92 ± 0.23 | 18.01 ± 1.56 | 22.15 ± 1.54 |
Juvenile stages | 28.2 ± 3.25 | 1.86 ± 0.21 | 20.15 ± 2.12 | 23.45 ± 2.35 |
Leaf Discs | 45.2 ± 2.32 | 1.98 ± 0.18 | 21.45 ± 2.36 | 24.56 ± 3.21 |
Concentration | Technical Replicates | Cq Mean ± S.D. | ||
---|---|---|---|---|
A | B | C | ||
5 ng/μL | 20.92 | 20.63 | 20.79 | 20.78 ± 0.145 |
1 ng/μL | 23.07 | 22.91 | 23.08 | 23.02 ± 0.095 |
200 pg/µL | 25.28 | 25.12 | 25.41 | 25.27 ± 0.090 |
40 pg/µL | 26.71 | 26.77 | 27.13 | 26.87 ± 0.227 |
8 pg/µL | 28.85 | 29.32 | 29.57 | 29.25 ± 0.366 |
1.6 pg/µL | 31.27 | 30.98 | 31.87 | 31.37 ± 0.454 |
0.32 pg/µL | 33.58 | 35.78 | 33.3 | 34.22 ± 1.358 |
0.064 pg/µL | 35.71 | 38.87 | 37.63 | 37.4 ± 1.592 |
0.0128 pg/µL | N.A. | N.A. | N.A. | - |
5.12 fg/µL | N.A. | N.A. | N.A. | - |
Repeatability | ||||||||
Test | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
A | 27.16 | 27.79 | 27.94 | 27.92 | 27.26 | 27.6 | 27.7 | 27.9 |
B | 27.64 | 27.46 | 27.43 | 27.27 | 27.35 | 27.41 | 27.56 | 27.13 |
C | 27.54 | 27.08 | 27.21 | 27.05 | 26.98 | 27.03 | 27.21 | 27.3 |
Cq Mean ± S.D. | 27.45 ± 0.253 | 27.44 ± 0.269 | 27.53 ± 0.156 | 27.41 ± 0.156 | 27.20 ± 0.262 | 27.35 ± 0.269 | 27.49 ± 0.247 | 27.44 ± 0.120 |
Reproducibility | ||||||||
Test | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
A | 27.51 | 27.19 | 27.1 | 27.09 | 27.22 | 27.04 | 27.31 | 27.33 |
B | 27.77 | 27.16 | 27.18 | 27.24 | 27.55 | 27.63 | 27.14 | 27.18 |
C | 27.52 | 27.13 | 27.12 | 27.54 | 27.86 | 27.57 | 27.76 | 27.4 |
Cq Mean ± S.D. | 27.6 ± 0.147 | 27.16 ± 0.030 | 27.13 ± 0.042 | 27.29 ± 0.229 | 27.54 ± 0.320 | 27.41 ± 0.325 | 27.40 ± 0.320 | 27.30 ± 0.112 |
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Rizzo, D.; Zubieta, C.G.; Moriconi, M.; Carli, M.; Marrucci, A.; Ranaldi, C.; Palmigiano, B.; Bartolini, L.; Pica, F.; Carbone, C.; et al. Streamlining the Identification of the Orange Spiny Whitefly, Aleurocanthus spiniferus (Hemiptera: Aleyrodidae), with Real-Time PCR Probe Technology. Agriculture 2025, 15, 414. https://doi.org/10.3390/agriculture15040414
Rizzo D, Zubieta CG, Moriconi M, Carli M, Marrucci A, Ranaldi C, Palmigiano B, Bartolini L, Pica F, Carbone C, et al. Streamlining the Identification of the Orange Spiny Whitefly, Aleurocanthus spiniferus (Hemiptera: Aleyrodidae), with Real-Time PCR Probe Technology. Agriculture. 2025; 15(4):414. https://doi.org/10.3390/agriculture15040414
Chicago/Turabian StyleRizzo, Domenico, Claudia Gabriela Zubieta, Michela Moriconi, Marco Carli, Andrea Marrucci, Chiara Ranaldi, Bruno Palmigiano, Linda Bartolini, Feliciana Pica, Carmela Carbone, and et al. 2025. "Streamlining the Identification of the Orange Spiny Whitefly, Aleurocanthus spiniferus (Hemiptera: Aleyrodidae), with Real-Time PCR Probe Technology" Agriculture 15, no. 4: 414. https://doi.org/10.3390/agriculture15040414
APA StyleRizzo, D., Zubieta, C. G., Moriconi, M., Carli, M., Marrucci, A., Ranaldi, C., Palmigiano, B., Bartolini, L., Pica, F., Carbone, C., Massimino Cocuzza, G. E., & Nugnes, F. (2025). Streamlining the Identification of the Orange Spiny Whitefly, Aleurocanthus spiniferus (Hemiptera: Aleyrodidae), with Real-Time PCR Probe Technology. Agriculture, 15(4), 414. https://doi.org/10.3390/agriculture15040414