Assay Optimization Can Equalize the Sensitivity of Real-Time PCR with ddPCR for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) in Bulk Samples

Helicoverpa armigera (Hübner) is one of the most important agricultural pests in the world. This historically Old World species was first reported in Brazil in 2013 and has since spread throughout much of South America and into the Caribbean. Throughout North America, H. armigera surveys are ongoing to detect any incursions. Each trap is capable of capturing hundreds of native Helicoverpa zea (Boddie). The two species cannot be separated without genitalic dissection or molecular methods. A ddPCR assay is currently used to screen large trap samples, but this equipment is relatively uncommon and expensive. Here, we optimized a newly designed assay for accurate and repeatable detection of H. armigera in bulk samples across both ddPCR and less costly, and more common, real-time PCR methods. Improvements over previously designed assays were sought through multiple means. Our results suggest bulk real-time PCR assays can be improved through changes in DNA extraction and purification, so that real-time PCR can be substituted for ddPCR in screening projects. While ddPCR remains a more sensitive method for detection of H. armigera in bulk samples, the improvements in assay design, DNA extraction, and purification presented here also enhance assay performance over previous protocols.

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Effects of DNA Extraction and Purification Methods on Real-Time Quantitative PCR Analysis of Roundup Ready Soybean

Journal of AOAC International ◽

10.1093/jaoac/92.4.1136 ◽

2009 ◽

Vol 92 (4) ◽

pp. 1136-1144 ◽

Cited By ~ 18

Author(s):

Tigst Demeke ◽

Indira Ratnayaka ◽

Anh Phan

Keyword(s):

Real Time ◽

Dna Extraction ◽

Quantitative Pcr ◽

Real Time Pcr ◽

Roundup Ready ◽

Dna Recovery ◽

Real Time Quantitative Pcr ◽

Extraction And Purification ◽

Purification Methods ◽

Accuracy And Repeatability

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

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Phylogeography of the Recent Expansion of Helicoverpa armigera (Lepidoptera: Noctuidae) in South America and the Caribbean Basin

Annals of the Entomological Society of America ◽

10.1093/aesa/saz019 ◽

2019 ◽

Vol 112 (4) ◽

pp. 388-401 ◽

Cited By ~ 7

Author(s):

Luke R Tembrock ◽

Alicia E Timm ◽

Frida A Zink ◽

Todd M Gilligan

Keyword(s):

South America ◽

Helicoverpa Armigera ◽

Helicoverpa Zea ◽

New World ◽

The United States ◽

Caribbean Basin ◽

Agricultural Pests ◽

Multiple Introductions ◽

Helicoverpa Armígera ◽

The Caribbean

Abstract The Old World bollworm, Helicoverpa armigera (Hübner), is one of the most destructive agricultural pests worldwide. It was first recorded in Brazil in 2013, yet despite this recent introduction, H. armigera has spread throughout much of Latin America. Where H. armigera has become established, it is displacing or hybridizing with the congeneric New World pest Helicoverpa zea. In addition to the adaptive qualities that make H. armigera a megapest, such as broad range pesticide resistance, the spread of H. armigera in the New World may have been hastened by multiple introductions into South America and/or the Caribbean. The recent expansion of the range of H. armigera into the New World is analyzed herein using mtDNA of samples from South America, the Caribbean Basin, and the Florida Peninsula. Phylogeographic analyses reveal that several haplotypes are nearly ubiquitous throughout the New World and native range of H. armigera, but several haplotypes have limited geographic distribution from which a secondary introduction with Euro-African origins into the New World is inferred. In addition, host–haplotype correlations were analyzed to see whether haplotypes might be restricted to certain crops. No specialization was found; however, some haplotypes had a broader host range than others. These results suggest that the dispersal of H. armigera in the New World is occurring from both natural migration and human-mediated introductions. As such, both means of introduction should be monitored to prevent the spread of H. armigera into areas such as the United States, Mexico, and Canada, where it is not yet established.

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Identification and Evaluation of Suitable Reference Genes for Normalization of MicroRNA Expression in Helicoverpa armigera (Lepidoptera: Noctuidae) Using Quantitative Real-Time PCR

Journal of Insect Science ◽

10.1093/jisesa/iex007 ◽

2017 ◽

Vol 17 (2) ◽

Cited By ~ 6

Author(s):

Yuhui Yang ◽

Zhen Li ◽

Jinjun Cao ◽

Yanrong Li ◽

Hui Li ◽

...

Keyword(s):

Real Time ◽

Helicoverpa Armigera ◽

Real Time Pcr ◽

Reference Genes ◽

Microrna Expression ◽

Quantitative Real Time Pcr ◽

Helicoverpa Armígera ◽

Suitable Reference ◽

Lepidoptera Noctuidae

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Real-Time PCR Analysis of Pyrethroid Resistance in Helicoverpa armigera from Turkey

Turkish Journal of Biochemistry ◽

10.5505/tjb.2014.50490 ◽

2014 ◽

Vol 39 (2) ◽

pp. 176-180 ◽

Cited By ~ 1

Author(s):

Metin Konus ◽

Sakine Ugurlu Karaagac ◽

Mesude Iscan

Keyword(s):

Real Time ◽

Helicoverpa Armigera ◽

Real Time Pcr ◽

Pyrethroid Resistance ◽

Pcr Analysis ◽

Helicoverpa Armígera

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Enrichment and DNA Extraction Protocols for the Simultaneous Detection of Salmonella and Listeria monocytogenes in Raw Sausage Meat with Multiplex Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-67.1.189 ◽

2004 ◽

Vol 67 (1) ◽

pp. 189-192 ◽

Cited By ~ 58

Author(s):

XIAOWEN WANG ◽

NARAYANAN JOTHIKUMAR ◽

MANSEL W. GRIFFITHS

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Meat Sample ◽

Extraction And Purification ◽

Pcr Products ◽

Novel Method ◽

Hlya Gene

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes, and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.

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