Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris
The application of quantitative PCR (qPCR) as a routine method to detect and enumerate <i>Paenibacillus larvae</i> in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of <i>Apis mellifera</i>....
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-11-01
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| Series: | Insects |
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| Online Access: | https://www.mdpi.com/2075-4450/9/4/165 |
| _version_ | 1818486145437138944 |
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| author | Franca Rossi Carmela Amadoro Addolorato Ruberto Luciano Ricchiuti |
| author_facet | Franca Rossi Carmela Amadoro Addolorato Ruberto Luciano Ricchiuti |
| author_sort | Franca Rossi |
| collection | DOAJ |
| description | The application of quantitative PCR (qPCR) as a routine method to detect and enumerate <i>Paenibacillus larvae</i> in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of <i>Apis mellifera</i>. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for <i>P. larvae</i> detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of <i>P. larvae</i> spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired <i>P. larvae</i> sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of a previously described method by shortening the forward primer, designing a new reverse primer and using more stringent amplification conditions. The new test allowed the detection of <i>P. larvae</i> spores in honey and hive debris down to 1 CFU/g. The qPCR test optimized in this study proved suitable for quantification and also for identification of field <i>P. larvae</i> strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of <i>P. larvae</i> in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures. |
| first_indexed | 2024-12-10T16:18:58Z |
| format | Article |
| id | doaj.art-447c57a8892c4d9fb6319834baf65082 |
| institution | Directory Open Access Journal |
| issn | 2075-4450 |
| language | English |
| last_indexed | 2024-12-10T16:18:58Z |
| publishDate | 2018-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Insects |
| spelling | doaj.art-447c57a8892c4d9fb6319834baf650822022-12-22T01:41:53ZengMDPI AGInsects2075-44502018-11-019416510.3390/insects9040165insects9040165Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive DebrisFranca Rossi0Carmela Amadoro1Addolorato Ruberto2Luciano Ricchiuti3Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario 1, 64100 Teramo, ItalyMedicine and Health Science Department “V. Tiberio”, University of Molise, Via de Santis, 86100 Campobasso, ItalyIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario 1, 64100 Teramo, ItalyIstituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario 1, 64100 Teramo, ItalyThe application of quantitative PCR (qPCR) as a routine method to detect and enumerate <i>Paenibacillus larvae</i> in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of <i>Apis mellifera</i>. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for <i>P. larvae</i> detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of <i>P. larvae</i> spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired <i>P. larvae</i> sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of a previously described method by shortening the forward primer, designing a new reverse primer and using more stringent amplification conditions. The new test allowed the detection of <i>P. larvae</i> spores in honey and hive debris down to 1 CFU/g. The qPCR test optimized in this study proved suitable for quantification and also for identification of field <i>P. larvae</i> strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of <i>P. larvae</i> in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.https://www.mdpi.com/2075-4450/9/4/165<i>Paenibacillus larvae</i>optimized qPCRquantificationhoneyhive debris |
| spellingShingle | Franca Rossi Carmela Amadoro Addolorato Ruberto Luciano Ricchiuti Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris Insects <i>Paenibacillus larvae</i> optimized qPCR quantification honey hive debris |
| title | Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris |
| title_full | Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris |
| title_fullStr | Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris |
| title_full_unstemmed | Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris |
| title_short | Evaluation of Quantitative PCR (qPCR) <i>Paenibacillus larvae</i> Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris |
| title_sort | evaluation of quantitative pcr qpcr i paenibacillus larvae i targeted assays and definition of optimal conditions for its detection quantification in honey and hive debris |
| topic | <i>Paenibacillus larvae</i> optimized qPCR quantification honey hive debris |
| url | https://www.mdpi.com/2075-4450/9/4/165 |
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