scholarly journals Development of a simultaneous amplification and testing (SAT) system based fluorescence real-time isothermal RNA amplification for rapid detection of Cronobacter spp. in powdered infant formula

2020 ◽  
Author(s):  
Riqin Wu ◽  
Xiaojiao Song ◽  
Xiaoping Gong ◽  
Gang Wang ◽  
Xinsheng Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Direct Detection ◽  
Detection System ◽  
Specific Method ◽  
Powdered Infant Formula ◽  
Infant Formulas ◽  
Rna Amplification

Abstract Contamination of Cronobacter spp. in powdered infant formulas is a severe food safety problem. The present study developed a rapid and sensitive Simultaneous Amplification and Testing (SAT) system for the detection of Cronobacter spp. in powdered infant formula. SAT detection system is based on fluorescence real-time isothermal RNA amplification and mainly includes pre-enrichment, RNA isolation and detection by fluorescence real-time RNA isothermal amplification. The amplification targets 16 s/23 s rRNA for the specific detection and rapid identification of Cronobacter spp. and can accurately detect viable strains in infant formulas and other food products. Here, six C. sakazakii strains and 25 references strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods like ISO-IDF 22964 and real-time PCR. The SAT assay was proved to be highly sensitive with a detection limit of 102 CFU/mL without pre-enrichment for powdered infant formula. After 3 h, 4 h and 8 h enrichment, the sensitivity was increased up to 100, 10− 1 and 10− 3 CFU/mL of Cronobacter spp., respectively. The SAT system including pre-enrichment performed for Cronobacter spp. detection was less than 4 h, dramatically shortened, in comparison to several days using standard culturing method and overnight using pre-enrichment real-time PCR method. And more importantly, the SAT assay can accurately distinguish viable strains from the dead one. Taken together, the SAT assay combined with pre-enrichment established in the present study should provide a rapid, sensitive, efficient and specific method for direct detection of Cronobacter spp. in powdered infant formula. Moreover, a full automatic food-borne pathogenic bacteria detector was developed based on the SAT assay.

Development of an Improved Protocol for the Isolation and Detection of Enterobacter sakazakii (Cronobacter) from Powdered Infant Formula

2010 ◽  
Vol 73 (6) ◽  
pp. 1016-1022 ◽  
Author(s):  
YI CHEN ◽  
KWANG-YONG SONG ◽  
ERIC W. BROWN ◽  
KEITH A. LAMPEL
Keyword(s):  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Powdered Infant Formula ◽  
Enterobacter Sakazakii ◽  
Biochemical Tests ◽  
The Real

Enterobacter sakazakii causes severe maladies and, in some cases, is fatal among infants. Powdered infant formula (PIF) contaminated with E. sakazakii has been documented as a potential cause of several outbreaks involving infants. This study describes the development of a method for the isolation and detection of E. sakazakii from PIF. It combines Taqman real-time PCR, Brilliance E. sakazakii and R&F chromogenic agars, and RAPID ID 32E biochemical tests. This method provides an expedient analysis within 1 to 2 days depending on the amount and stress status of E. sakazakii organisms and competing microorganisms in PIF. The real-time PCR has bifunctional applications, including both screening and culture confirmation of E. sakazakii.

Direct Real-Time PCR with Ethidium Monoazide: A Method for the Rapid Detection of Viable Cronobacter sakazakii in Powdered Infant Formula

2012 ◽  
Vol 75 (9) ◽  
pp. 1572-1579 ◽  
Author(s):  
JUN-ICHI MINAMI ◽  
TAKASHI SOEJIMA ◽  
TOMOKO YAESHIMA ◽  
KEIJI IWATSUKI
Keyword(s):  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Powdered Infant Formula ◽  
Cronobacter Sakazakii ◽  
Ethidium Monoazide ◽  
Rapid Assay ◽  
Negative Results ◽  
Current Detection ◽  
Positive Results

The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMADqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.

Real-time PCR targeting OmpA gene for detection of Cronobacter spp. in powdered infant formula

2013 ◽  
Vol 22 (2) ◽  
pp. 309-313 ◽  
Author(s):  
Xiaohui Dong ◽  
Qingping Wu ◽  
Kui Wu ◽  
Jumei Zhang
Keyword(s):  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Powdered Infant Formula ◽  
Ompa Gene ◽  
Pcr Targeting

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system

2004 ◽  
Vol 30 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Sani Hussein Aliyu ◽  
Muktar Hassan Aliyu ◽  
Hamisu M Salihu ◽  
Surendra Parmar ◽  
Hamid Jalal ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Detection System ◽  
Hepatitis B Virus Dna ◽  
B Virus

scholarly journals Direct detection of microorganisms responsible for the sexually transmitted infection on the mobile real-time PCR device without treating in advance

2021 ◽  
Author(s):  
Masaaki Muraoka ◽  
Kazunori Sohma ◽  
Osamu Kawaguchi ◽  
Mikio Mizukoshi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Treponema Pallidum ◽  
Nucleic Acid Amplification ◽  
Direct Pcr ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Ct Value

ABSTRACTAs WHO reported, four curable STIs-chlamydia, gonorrhoea, syphilis and trichomoniasis occur more than 1 million per each day globally almond 2016. For this reason, it is important to control these STIs, one of which is “to detect”. The general methods in order to detect STIs are nucleic acid amplification tests (NAATs). One of the reasons why NAATs are utilized in many tests is that it is possibly to be more sensitive than other test. However, there needs to treat extraction of nucleic acids in advance and amplify specific regions by NAATs, and hence it must take much labour and much time. In this work, for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP) which is each etiological agent of chlamydia, gonorrhoea and syphilis, we evaluate and propose “quicker and simpler” NAATs. Specifically, utilizing mobile real-time PCR device “PCR1100” and PCR reagent kit “KAPA3G Plant PCR Kit”, it was considered whether real-time direct PCR could be performed or not without treating DNA extraction in advance so-called “direct”.As a result, firstly, we established that real-time direct PCR could be performed in all of CT, NG, and TP, and moreover, each Ct value correlated with the concentration of each organism similarly to detection of genome DNA (each correlation coefficient R2 > 0.95). Moreover, each assay demonstrated a limit of detection (LOD) of the follows; CT was 10^0.86 = 7.24 IFU/reaction, NG was 10^-0.19 = 0.65 CFU/reaction, and TP was 10^1.4 = 25.1 organisms/reaction. However, it appeared the sensitivity was a little low, especially for CT and TP.Secondly, we found that even as without treating sample in advance, the time of detection was required more less 15 minutes at any of case, which was very quick compared with other current methods for real-time PCR. Additionally, compared with other commercial devices, it was easier to operate the PCR1100 device, for example, start, analysis of Ct value.In conclusion, the present study has demonstrated that it is possible for real-time direct PCR to perform with combination of the PCR1100 device and the PCR reagent kit in 3 kinds of microorganisms-CT, NG and TP. Furthermore, we propose “quicker and simpler” methods for NAATs, which it would not take labour and time. Further studies are needed in order to contribute to control STIs.

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