SYBR Green real-time qPCR method: Diagnose drowning more rapidly and accurately

ABSTRACT A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).

Download Full-text

IDENTIFIKASI ZIGOSITAS IKAN LELE (Clarias gariepinus) TRANSGENIK F-2 YANG MEMBAWA GEN HORMON (PhGH) DENGAN MENGGUNAKAN METODE REALTIME-qPCR

Jurnal Riset Akuakultur ◽

10.15578/jra.11.1.2016.39-46 ◽

2016 ◽

Vol 11 (1) ◽

pp. 39

Author(s):

Huria Marnis ◽

Bambang Iswanto ◽

Romy Suprapto ◽

Imron Imron ◽

Raden Roro Sri Pudji Sinarni Dewi

Keyword(s):

Real Time ◽

Clarias Gariepinus ◽

Fish Farming ◽

Progeny Test ◽

African Catfish ◽

Mendelian Segregation ◽

Qpcr Method ◽

Pcr Method ◽

Real Time Qpcr ◽

Transgene Transmission

Produktivitas ikan budidaya dapat ditingkatkan melalui teknologi transgenesis. Populasi ikan lele transgenik cepat tumbuh telah dihasilkan dan karakter biologisnya telah diketahui. Namun informasi zigositas ikan lele transgenik perlu ditelaah lebih lanjut. Penelitian ini bertujuan untuk mengidentifikasi zigositas ikan lele transgenik F-2. Zigositas ikan lele transgenik diidentifikasi dengan menggunakan metode real-time qPCR (RT-qPCR) dan uji progeni. Identifikasi zigositas melalui uji progeni, dilakukan dengan mendeteksi transgen (PhGH) pada individu-individu F-3 hasil persilangan transgenik F-2 dengan non-transgenik. Hasil penelitian menunjukkan bahwa zigositas pada ikan lele transgenik F-2 dapat diidentifikasi dengan menggunakan metode RT-qPCR. Semua ikan transgenik F-2 adalah heterozigot, dengan nilai 2-Ct yang hampir sama tiap individu F-2, yaitu berkisar 0,80-0,99. Identifikasi zigositas dengan metode RT-qPCR menunjukkan hasil yang sama dengan uji progeni, semua transgenik F-2 tidak menghasilkan 100% anakan F-3 positif transgen. Pada uji progeni, transmisi transgen pada penelitian ini tidak mengikuti hukum segregasi Mendel, dengan kisaran sebesar 5%-40%.Fish farming productivity can be increased by transgenesis technology. On the previous study, transgenic African catfish population fast growing has been produced and its biological characters has been known. However information of transgenic zygosity of catfish should be examined. The aim of this study was to identify the zygosity of F-2 transgenic African catfish. The zygosity of F-2 transgenic was identified by real time-qPCR (RT-qPCR) method and progeny test. Further, identification of zygosity F-2 transgenic African catfish was confirmed by progeny test, while F-2 transgenic African catfish was mated with non-transgenic. Identification of zygosity F-2 transgenic was conducted by detection PhGH gene (transgene) in F-3 transgenic African catfish population. Transgene transmission was evaluated by PCR method. The result showed that the zygosity F-2 transgenic African catfish could be identified by RT-qPCR method. All F-2 transgenic African catfish were heterozygous, where as the 2-Ct value was almost same for all individual, which ranges from 0.80 to 0.99. The result of zygosity identification using RT-qPCR method was as same as that of progeny test. In the progeny test, transgene transmission in this study was non-Mendelian segregation, with ranges of 5%-40%.

Download Full-text

Application of a real-time qPCR method to measure the methanogen concentration during anaerobic digestion as an indicator of biogas production capacity

Journal of Environmental Management ◽

10.1016/j.jenvman.2012.07.021 ◽

2012 ◽

Vol 111 ◽

pp. 173-177 ◽

Cited By ~ 49

Author(s):

Deborah Traversi ◽

Silvia Villa ◽

Eugenio Lorenzi ◽

Raffaella Degan ◽

Giorgio Gilli

Keyword(s):

Anaerobic Digestion ◽

Real Time ◽

Biogas Production ◽

Production Capacity ◽

Qpcr Method ◽

Real Time Qpcr

Download Full-text

Author Correction: A simple and robust real-time qPCR method for the detection of PIK3CA mutations

Scientific Reports ◽

10.1038/s41598-021-95597-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Virginia Alvarez-Garcia ◽

Clare Bartos ◽

Ieva Keraite ◽

Urmi Trivedi ◽

Paul M. Brennan ◽

...

Keyword(s):

Real Time ◽

Pik3ca Mutations ◽

Qpcr Method ◽

Real Time Qpcr

Download Full-text

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

10.21203/rs.3.rs-37815/v1 ◽

2020 ◽

Author(s):

Wallop Jakkul ◽

Kittipong Chaisiri ◽

Naowarat Saralamba ◽

Yanin Limpanont ◽

Sirilak Dusitsittipon ◽

...

Keyword(s):

Intermediate Host ◽

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Eosinophilic Meningitis ◽

Quantitative Real Time Pcr ◽

The Third ◽

Snail Intermediate Host ◽

Third Stage ◽

Qpcr Method

Abstract Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentification often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specific primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal fluid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the field.Results: The newly designed primers were highly specific and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10−4 ng of genomic DNA from one larva and facilitated the specific detection and identification of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identification and has sufficient sensitivity and specificity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and definitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients with eosinophilic meningitis to assess the usefulness of the method for clinical diagnosis.

Download Full-text

Establishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmealestablishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmeal

10.21203/rs.3.rs-78082/v1 ◽

2020 ◽

Author(s):

Jinghua Ruan ◽

Wujun Wang ◽

Tiying Zhang ◽

Teng Zheng ◽

Jing Zheng ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Bacterial Species ◽

High Specificity ◽

Clinical Samples ◽

Amplification Efficiency ◽

Salmonella Spp ◽

Specificity And Sensitivity ◽

Qpcr Method ◽

Real Time Qpcr

Abstract Salmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratiafonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/SL and 145 copies/SL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

Download Full-text

Comparison of quantitative methods based on SYBR Green real-time qPCR to estimate pork meat adulteration in processed beef products

Food Chemistry ◽

10.1016/j.foodchem.2018.06.141 ◽

2018 ◽

Vol 269 ◽

pp. 549-558 ◽

Cited By ~ 11

Author(s):

Tae Sun Kang ◽

Takuji Tanaka

Keyword(s):

Real Time ◽

Quantitative Methods ◽

Sybr Green ◽

Pork Meat ◽

Beef Products ◽

Real Time Qpcr

Download Full-text

Establishment of a duplex real-time qPCR method for detection of Salmonella spp. and Serratia fonticola in fishmeal

AMB Express ◽

10.1186/s13568-020-01144-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jinghua Ruan ◽

Wujun Wang ◽

Tiyin Zhang ◽

Teng Zheng ◽

Jing Zheng ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Bacterial Species ◽

Bacterial Pathogen ◽

High Specificity ◽

Amplification Efficiency ◽

Salmonella Spp ◽

Specificity And Sensitivity ◽

Qpcr Method ◽

Real Time Qpcr

AbstractSalmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratia fonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of fishmeal was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

Download Full-text