Multiplex Real-Time PCR with HRM for Detection of <i>Lactobacillus </i>sakei and <i>Lactobacillus curvatus </i>in Food Samples

Kurbakov, K. A.; Коноров, Е. А.; Minaev, M. Yu.; Кузнецова, О. А.

doi:10.17113/ftb.57.01.19.5983

Cited by 10 publications

(5 citation statements)

References 33 publications

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“…The melting temperature obtained by Bar-HRM analysis remained unchanged in the fourth round of DNA template dilution ( S3 Appendix ). This finding was consistent with previous works on the stability of HRM results showing that the melting temperature did not vary within four logarithms of the initial concentration [ 23 , 37 ]. Moreover, the use of the rbc L region for species differentiation at the genus level has been revealed [ 38 ].…”

Section: Discussionsupporting

confidence: 93%

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, “Kho-Khlan”

et al. 2022

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The pain relief formula “Ya Pa Som Kho-Khlan (YPSKK)” or “ยาผสมโคคลาน” in Thai is officially recorded in the Natural List of Essential Medicines (NLEM) of Thailand. The main component is Mallotus repandus (Willd.) Müll. Arg.; however, Anamirta cocculus (L.) Wight & Arn and Croton caudatus Gleiseler share the same common name: “Kho-Khlan”. Confused usage of A. cocculus or C. caudatus can have effects via toxicity or unsuccessful treatment. This study aimed to combine a high-performance thin-layer chromatography (HPTLC) technique and DNA barcoding coupled with high-resolution melting (Bar-HRM) to differentiate M. repandus from the other two species. The M. repandus extract exhibited a distinct HPTLC profile that could be used to differentiate it from the others. DNA barcodes of the rbcL, matK, ITS and psbA-trnH intergenic spacer regions of all the plants were established to assist HPTLC analysis. The rbcL region was selected for Bar-HRM analysis. PCR amplification was performed to obtain 102 bp amplicons encompassing nine polymorphic nucleotides. The amplicons were subjected to HRM analysis to obtain melting curve profiles. The melting temperatures (Tm) of authentic A. cocculus (A), C. caudatus (C) and M. repandus (M) were separated at 82.03±0.09°C, 80.93±0.04°C and 80.05±0.07°C, respectively. The protocol was applied to test crude drugs (CD1-6). The HPTLC profiles of CD2-6 showed distinct bands of M. repandus, while CD1 showed unclear band results. The Bar-HRM method was applied to assist the HPTLC and indicated that CD1 was C. caudatus. While ambiguous melting curves from the laboratory-made formulae were obtained, HPTLC analysis helped reveal distinct patterns for the identification of the plant species. The combination of HPTLC and Bar-HRM analysis could be a tool for confirming the identities of plant species sharing the same name, especially for those whose sources are multiple and difficult to identify by either chemical or DNA techniques.

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Section: Discussionsupporting

confidence: 93%

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, “Kho-Khlan”

et al. 2022

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“…Also, droplet digital PCR, which is based on the fractionation of the sample into 20,000 droplets so that PCR amplification of the template molecules occurs in each individual droplet, allows for the absolute quantification of targets with a high sensitivity, and without the use of the standard curve [76]. Speciesand even strain-specific primers and probes have been designed to allow the real-time PCR HRM-and ddPCR-based identification of technological and probiotic microorganisms and the authentication of probiotic and fermented foods [77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92].…”

Section: Real-time Pcr-based Methodsmentioning

confidence: 99%

Recent and Advanced DNA-Based Technologies for the Authentication of Probiotic, Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) Fermented Foods and Beverages

Fusco,

Fanelli,

Chieffi

2023

Foods

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The authenticity of probiotic products and fermented foods and beverages that have the status of protected designation of origin (PDO) or geographical indication (PGI) can be assessed via numerous methods. DNA-based technologies have emerged in recent decades as valuable tools to achieve food authentication, and advanced DNA-based methods and platforms are being developed. The present review focuses on the recent and advanced DNA-based techniques for the authentication of probiotic, PDO and PGI fermented foods and beverages. Moreover, the most promising DNA-based detection tools are presented. Strain- and species-specific DNA-based markers of microorganisms used as starter cultures or (probiotic) adjuncts for the production of probiotic and fermented food and beverages have been exploited for valuable authentication in several detection methods. Among the available technologies, propidium monoazide (PMA) real-time polymerase chain reaction (PCR)-based technologies allow for the on-time quantitative detection of viable microbes. DNA-based lab-on-a-chips are promising devices that can be used for the on-site and on-time quantitative detection of microorganisms. PCR-DGGE and metagenomics, even combined with the use of PMA, are valuable tools allowing for the fingerprinting of the microbial communities, which characterize PDO and PGI fermented foods and beverages, and they are necessary for authentication besides permitting the detection of extra or mislabeled species in probiotic products. These methods, in relation to the authentication of probiotic foods and beverages, need to be used in combination with PMA, culturomics or flow cytometry to allow for the enumeration of viable microorganisms.

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“…Real-time assays are useful for distinguishing serotypes within a particular microbial population [73], diagnosing pathogens in clinical samples [74,75] and bacteria (viruses, bacteria, fungi, protozoa or toxins produced by them that cause diseases) used as biological warfare agents [76]. Despite these, molecular techniques including PCR do not provide any information about AMR among pathogens [77].…”

Section: Real-time Pcrmentioning

confidence: 99%

Fast Track Diagnostic Tools for Clinical Management of Sepsis: Paradigm Shift from Conventional to Advanced Methods

et al. 2023

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Sepsis is one of the deadliest disorders in the new century due to specific limitations in early and differential diagnosis. Moreover, antimicrobial resistance (AMR) is becoming the dominant threat to human health globally. The only way to encounter the spread and emergence of AMR is through the active detection and identification of the pathogen along with the quantification of resistance. For better management of such disease, there is an essential requirement to approach many suitable diagnostic techniques for the proper administration of antibiotics and elimination of these infectious diseases. The current method employed for the diagnosis of sepsis relies on the conventional culture of blood suspected infection. However, this method is more time consuming and generates results that are false negative in the case of antibiotic pretreated samples as well as slow-growing microbes. In comparison to the conventional method, modern methods are capable of analyzing blood samples, obtaining accurate results from the suspicious patient of sepsis, and giving all the necessary information to identify the pathogens as well as AMR in a short period. The present review is intended to highlight the culture shift from conventional to modern and advanced technologies including their limitations for the proper and prompt diagnosing of bloodstream infections and AMR detection.

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Multiplex Real-Time PCR with HRM for Detection of Lactobacillus sakei and Lactobacillus curvatus in Food Samples

Cited by 10 publications

References 33 publications

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, “Kho-Khlan”

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, “Kho-Khlan”

Recent and Advanced DNA-Based Technologies for the Authentication of Probiotic, Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) Fermented Foods and Beverages

Fast Track Diagnostic Tools for Clinical Management of Sepsis: Paradigm Shift from Conventional to Advanced Methods

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