Rapid diagnosis of human adenovirus B,�C�and�E in the respiratory tract using multiplex quantitative polymerase chain reaction

Abstract Background: Recombinase-aided amplification(RAA) is a new, simple, and ultrafast isothermal molecular diagnostic technique performed within 30min at 39°C–42°C.In this study, we evaluated the clinical performance of four duplex RAA kits for hepatitis B virus(HBV), human adenovirus 3(HAdV3), human adenovirus 7(HAdV7), and Bordetella pertussis and one duplex reverse-transcription RAA (RT-RAA) kit for respiratory syncytial virus (RSV).Methods: A total of 392 sera and 374 respiratory tract samples were collected from five institutions in four China regions. Each RAA kit’s sensitivity and specificity were compared with those of real-time quantitative polymerase chain reaction(qPCR),real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR), or sequencing. Results: Compared with qPCR or qRT-PCR, the sensitivities of HBV RAA,RSV RT-RAA, and B.pertussis RAA were 97.55%,96.67%, and 100%,respectively,and all of the specificities were 100%.The total coincidence rates were 97.78%(383/392,95%CI:95.63%–98.85%),97.70%(212/217, 95%CI:94.57%–99.16%), and 100%(60/60,95%CI:92.80%–100%),respectively.The Kappa values were 0.977,0.947, and 1,respectively(P<0.05).Regarding the sequencing, the sensitivities of HAdV3 RAA and HAdV7 RAA were 100% and 97.37%, respectively,and all specificities were 100%.The total coincidence rates were 100%(97/97,95%CI:91.58%–100%) and 98.97%(96/97,95%CI:94.39%–99.82%),and the Kappa values were 1 and 0.978 (P<0.05),respectively.Conclusions: With comparable clinical performance, these RAA kits are suitable assays for rapidly detecting pathogens in resource-limited laboratories.

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MMP1 is a Promiseful Prognostic Biomarker and Correlating with Immune Infiltrates in Hepatocellular Carcinoma

10.21203/rs.3.rs-547264/v1 ◽

2021 ◽

Author(s):

Lei Dai ◽

Joseph Muggnyi ◽

xingchen cai ◽

shuqi mao ◽

tongyue zhang ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Clinical Performance ◽

Quantitative Polymerase Chain Reaction ◽

Human Adenovirus ◽

Molecular Diagnostic ◽

Chain Reaction ◽

Qrt Pcr ◽

Polymerase Chain

Abstract Background: Recombinase-aided amplification(RAA) is a new, simple, and ultrafast isothermal molecular diagnostic technique performed within 30min at 39°C–42°C.In this study, we evaluated the clinical performance of four duplex RAA kits for hepatitis B virus(HBV), human adenovirus 3(HAdV3), human adenovirus 7(HAdV7), and Bordetella pertussis and one duplex reverse-transcription RAA (RT-RAA) kit for respiratory syncytial virus (RSV).Methods: A total of 392 sera and 374 respiratory tract samples were collected from five institutions in four China regions. Each RAA kit’s sensitivity and specificity were compared with those of real-time quantitative polymerase chain reaction(qPCR),real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR), or sequencing. Results: Compared with qPCR or qRT-PCR, the sensitivities of HBV RAA,RSV RT-RAA, and B.pertussis RAA were 97.55%,96.67%, and 100%,respectively,and all of the specificities were 100%.The total coincidence rates were 97.78%(383/392,95%CI:95.63%–98.85%),97.70%(212/217, 95%CI:94.57%–99.16%), and 100%(60/60,95%CI:92.80%–100%),respectively. The Kappa values were 0.977,0.947, and 1,respectively(P<0.05).Regarding the sequencing, the sensitivities of HAdV3 RAA and HAdV7 RAA were 100% and 97.37%, respectively, and all specificities were 100%.The total coincidence rates were 100%(97/97,95%CI:91.58%–100%) and 98.97%(96/97,95%CI:94.39%–99.82%),and the Kappa values were 1 and 0.978 (P<0.05),respectively.Conclusions: With comparable clinical performance, these RAA kits are suitable assays for rapidly detecting pathogens in resource-limited laboratories.

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AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

FLORA AND FAUNA ◽

10.33451/florafauna.v23i1pp25-36 ◽

2017 ◽

Vol 23 (1) ◽

Author(s):

N.NANDHA KUMAR ◽

K. SOURIANATHA SUNDARAM ◽

D. SUDHAKAR ◽

K.K. KUMAR

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Polymerase Chain Reaction ◽

Proteinase K ◽

Chain Reaction ◽

Banana Plant ◽

High Molecular Weight Dna ◽

Musa Spp ◽

Leaf Tissues ◽

Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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