Nucleic Acid Amplification Techniques For Respiratory Virus Detection

Catalogue 1 Introduction: The Type Of Nucleic Acid Amplification Techniques 2 Example For Respiratory

Most commonly used Techniques 1 PCR RT-PCR HDA TMA Nucleic Acid Amplification Techniques MLPA

1 Advantages and disadvantages PCR RT-PCR Easy to popularize in surveillance : Operator and

Branch research project of China National Science and Technology Major Project 2 Chinese Center

Example of Respiratory Virus Surveillance 2 Epidemiology of Respiratory Viruses in ARTI in Jinan

Procedure 2 1 Collection Upper ARTIs： Nasal ＆Throat Swabs+ Serum Lower ARTIs： Bronchoalveolar lavage

2 Result 1 v. Adult Upper ARTIs: 596 patients, 38. 76% of them positive

2 Articles published after project Q 2 IF: 3. 064 SMK 5404

3 Introspection and assumption Introspection after learned SMK 5404 Sensitivity Main technical problems in

3 Sensitivity Adult Upper ARTIs (July 2009 -- June 2010) Group Positive Rate Amount

Speed And Operational complexity 3 Technique Operation Time Running Time Of Instrument PCR 30

Question and Answer Question：What’s the differences between “RT-PCR” and “Real-Time” PCR? Answer: RT-PCR is

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Nucleic Acid Amplification Techniques For Respiratory Virus Detection Tong Jiabei GS 56087 18/12/2019

Catalogue 1 Introduction: The Type Of Nucleic Acid Amplification Techniques 2 Example For Respiratory Virus Surveillance 3 Introspection and assumption: Comparison of different technologies SMK 5404

Most commonly used Techniques 1 PCR RT-PCR HDA TMA Nucleic Acid Amplification Techniques MLPA Real-Time PCR RCA NASBA LAMP SDA SMK 5404

1 Advantages and disadvantages PCR RT-PCR Easy to popularize in surveillance : Operator and Equipment Long time, low sensitivity, low specificity Real-Time PCR Golden standard for many virus detection, Shorter time, high sensitivity, high specificity Higher cost, high -level equipment Fastest, high sensitivity, high specificity , low -level equipment LAMP Short fragment, aerosol contamination, reaction system/primer designing is complicate. SMK 5404

Branch research project of China National Science and Technology Major Project 2 Chinese Center for Disease Control and Prevention Peking Union Medical College Our group Adults: Upper ARTIs Children: Upper and Lower ARTIs 14 years old body temperature≥ 38℃ July 2009 -- June 2010 Research groups out of disease control system SMK 5404

Example of Respiratory Virus Surveillance 2 Epidemiology of Respiratory Viruses in ARTI in Jinan v. Virus Species HBo. V, HCo. V, IFVs, HRV， RSV, EV， SV PIV， HMPV， ADV v. Aim Investigate the viral aetiology in adults/children patients with Acute Respiratory Tract Infections in Jinan v. Technique Multiplex PCR ； Multiplex Reverse. Transcription PCR SMK 5404

Procedure 2 1 Collection Upper ARTIs： Nasal ＆Throat Swabs+ Serum Lower ARTIs： Bronchoalveolar lavage fluid+ Serum 2 Storage Every sample transport from hospital within 6 h, test/store in 80℃(3 tubes 300 ml) within 12 h. 3 Multiplex PCR/RT-PCR Every 3 -4 pairs of primers in a reaction system. Result was detected by agarose gel electrophoresis 4 Result ＆ Transport Analyze result and information recorded into the system. Transport to Bei Jing institution every 3 month. Patient information SMK 5404

2 Result 1 v. Adult Upper ARTIs: 596 patients, 38. 76% of them positive for viruses. v. IFV 20. 81%, HBo. V 8. 22%, HRV 5. 03%, EV 3. 69%, RSV 2. 52%, PIV 0. 84%, HMPV 0. 84%, ADV 0. 84% and HCo. V 0. 67% 2 v. Child: v 397 Upper ARTIs, 47. 10% positive for viruses, coinfection rate 6. 05%. v 323 Lower ARTIs, 66. 57% positive for viruses, coinfection rate 25. 98%. v. Most prevalent viruses: RV 19. 72%, PIV 17. 78%, and IFV 13. 9%. 3 v. Investigated the viral etiology in different month during July 2009 to June 2010 SMK 5404

2 Articles published after project Q 2 IF: 3. 064 SMK 5404

2 Adult: Upper ARTIs SMK 5404

2 Child: Upper and Lower ARTIs SMK 5404

3 Introspection and assumption Introspection after learned SMK 5404 Sensitivity Main technical problems in this Surveillance work Speed Operational Complexity Compare with Real-time PCR and LAMP SMK 5404

3 Sensitivity Adult Upper ARTIs (July 2009 -- June 2010) Group Positive Rate Amount of sample Beijing ≈56% ≈1900 Experiment/ Transport Protocol Operator Hospital Skilled Same Jinan 38. 76% 596 Skilled /New Maybe mistakes in made us get too little nucleic acid as PCR template SMK 5404

Speed And Operational complexity 3 Technique Operation Time Running Time Of Instrument PCR 30 -50 min 3 -3. 5 h Yes Real-Time PCR 15 -30 min 2 -3 h No LAMP 15 -30 min 15 -60 min No Electrophoresis Amount Of Sample 80 SMK 5404

Question and Answer Question：What’s the differences between “RT-PCR” and “Real-Time” PCR? Answer: RT-PCR is used to reverse transcript the RNA to c. DNA and then amplify it, Real-Time PCR used to detect, characterize and quantify nucleic acids for numerous applications. RT-PCR: Abbreviation for "Reverse Transcription" PCR , it is an laboratory technique combining reverse transcription of RNA into DNA (c. DNA) by reverse transcriptase enzyme and amplification of specific DNA targets using polymerase chain reaction (PCR). Real-Time PCR: Also named q. PCR or Quantitative PCR, it is a technique used to monitor the progress of a PCR reaction in real time, a relatively small amount of PCR product (DNA, c. DNA or RNA) can be quantified. Technical Characteristics of Real-Time: Add dye/probe group, Monitor the entire PCR process, Record Ct value and the standard curve. SMK 5404