Molecular approach for insect detection in feed and food: the case of Gryllodes sigillatus

AbstractThe production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.

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Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

Anti-Infective Agents ◽

10.2174/2211352518999200629165108 ◽

2020 ◽

Vol 18 ◽

Author(s):

Pegah Shakib ◽

Mohammad Reza Zolfaghari

Keyword(s):

Streptococcus Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Cross Sectional Study ◽

Laboratory Culture ◽

Cross Sectional ◽

Negative Results ◽

False Negative Results ◽

Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2013.06.024 ◽

2013 ◽

Vol 46 (15) ◽

pp. 1566-1571 ◽

Cited By ~ 10

Author(s):

Weidong Zheng ◽

Yuwei Di ◽

Yinghong Liu ◽

Ge Huang ◽

Youwei Zheng ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

Pcr Method

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MGB-based one-step multiplex real-time PCR method for rapid detection of HPV

Cancer Biomarkers ◽

10.3233/cbm-130298 ◽

2013 ◽

Vol 12 (3) ◽

pp. 107-113 ◽

Cited By ~ 1

Author(s):

Jie Huang ◽

Chun Gao ◽

Xilai Ding ◽

Shoufang Qu ◽

Licheng Liu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

One Step ◽

Pcr Method

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Rapid concentration and sensitive detection of hookworm ova from wastewater matrices using a real-time PCR method

Experimental Parasitology ◽

10.1016/j.exppara.2015.08.009 ◽

2015 ◽

Vol 159 ◽

pp. 5-12 ◽

Cited By ~ 17

Author(s):

P. Gyawali ◽

J.P.S. Sidhu ◽

W. Ahmed ◽

P. Jagals ◽

S. Toze

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sensitive Detection ◽

Pcr Method ◽

Hookworm Ova

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Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-013-7125-5 ◽

2013 ◽

Vol 405 (21) ◽

pp. 6831-6844 ◽

Cited By ~ 36

Author(s):

Geoffrey Cottenet ◽

Carine Blancpain ◽

Véronique Sonnard ◽

Poh Fong Chuah

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Pcr Method ◽

Development And Validation

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Real-time PCR method for identification of Asian populations in forensic casework

Legal Medicine ◽

10.1016/j.legalmed.2009.02.011 ◽

2009 ◽

Vol 11 ◽

pp. S106-S108 ◽

Cited By ~ 1

Author(s):

Tomoharu Tokutomi ◽

Yuzo Takada ◽

Takako Murayama ◽

Masahiro Mukaida ◽

Jun Kanetake

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Forensic Casework ◽

Asian Populations ◽

Pcr Method

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A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product

Biologicals ◽

10.1016/j.biologicals.2021.07.006 ◽

2021 ◽

Author(s):

V. Becherucci ◽

L. Curini ◽

R. Ceccantini ◽

S. Bisin ◽

V. Gori ◽

...

Keyword(s):

Medicinal Product ◽

Real Time ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Real Time Pcr ◽

Advanced Therapy Medicinal Product ◽

Human Mesenchymal Stromal Cells ◽

Mycoplasma Detection ◽

Advanced Therapy ◽

Pcr Method

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Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.20-1006 ◽

2021 ◽

Author(s):

Aymen Abdelhaleem ◽

Nabil Dhayhi ◽

Mohamed Salih Mahfouz ◽

Ommer Daffalla ◽

Mansour Mubarki ◽

...

Keyword(s):

Saudi Arabia ◽

Visceral Leishmaniasis ◽

Real Time ◽

Real Time Pcr ◽

Leishmania Donovani ◽

Target Genes ◽

Sample Volume ◽

Pcr Products ◽

Polymerase Chain ◽

Robust Evidence

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

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