TITLE: VPH_AUTOGENE-COVID_Diagnosis_Protocol_XPRIZE
AUTHORS:  Dr Vikas Pandey, Dr Saurabh Singh, Shruti Ahuja, Avishek Dev, Manu Kadyan, Mukesh Pandit, Amir Khan, Sambit Dam, Tanish Garg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Valetude Primus Healthcare (VPH) is a healthcare spinoff from IIT Delhi, India. VPH has developed an automated portable device, called AutoGene-COVID, to detect SARS COV 2 infection from a patient’s nasopharyngeal, oropharyngeal, nasal swab, or saliva sample within 2 hours. The one-step rapid process includes RNA extraction and purification using magnetic capture (< 30 minutes) followed by the confirmation of COVID-19 infection using reverse transcription-polymerase chain reaction (1 hour). The automated system comprises of an ingeniously designed single-use cartridge that has pre-filled reagents for RNA extraction and RT-PCR. The qualitative RT-PCR test detects the presence of Nucleocapsid genes N1, N2, and N3 and RNAse P gene by SYBR Green I based fluorescence. The over-all test costs less than $13.29/sample with sensitivity and specificity of 100% and a limit of detection of 10 copies/µL. The automation of the test ensures a contained system and reduces the risk of infection to the user. It obviates the need for infrastructure and trained personnel for the operation of the device. The battery-operated system is a portable device and can also be deployed in resource-poor settings. </div></div>

[STEPS]
?. [Sample Collection]
Collect nasopharyngeal or oropharyngeal samples from patients at a healthcare facility using a flocked tapered nylon swab. For specimen collection of nasal swabs, follow the CDC Swab Collection Guidelines and swab manufacturers' recommendations. Tilt the patient’s head back 70 degrees. While gently rotating the swab, insert swab less than one inch (about 2 cm) into the nostril (until resistance is met at turbinates). Rotate the swab several times against the nasal wall and repeat in other nostril using the same swab.
?. [Sample Collection]
Place and soak the Patient Swab in the VTM (Viral transport medium) vial. Rotate and stir the swab up and down for  .
?. [Sample Collection]
Scan the barcode on the cartridge and fill the patient's details in the device.
?. [RNA extarction using magnetic capture]
Transfer  from the VTM tube in the reaction slot of the cartridge using a micropipette. Place the cartridge in the device. Select the slots with cartridges and click on the 'RUN' option.
200 µl
The device can accommodate 10 samples at the same time.
?. [RNA extarction using magnetic capture]
In the cartridge,   of lysis buffer is pumped in the reaction slot and incubated for  . The composition of the lysis buffer is detailed below:  Guanidine thiocyanate,   Tris-HCl,   EDTA,   Triton-X, and    Proteinase-K.
200 µl
?. [RNA extarction using magnetic capture]
Binding buffer (Isopropanol) and   of magnetic beads are pumped in the reaction slot and incubated for .
265 µl
10 µl
?. [RNA extarction using magnetic capture]
An external magnetic field is applied to the reaction slot to separate the magnetic beads from the solution. The magnetic beads are washed twice and RNA bound to the beads are eluted in  elution buffer. Reagents:Wash buffer 1:   Nuclease free waterElution Buffer:    Tris-HCl,  ,
50 µl
500 µl
750 µl
?. [Reverse transcription PCR]
eluted RNA is transferred to PCR Slots 1, 2, 3, and 4. The PCR Slots are pre-filled with primers and PCR master mix.     AB1SuperScript® III RT/Platinum® Taq Mix0.5 μl22X SYBR® Green Reaction Mix12.5 μl3Forward Primer0.125 μl4Reverse primer0.125 μl5Nuclease free water1.75 μl6Extracted RNA10  μl7Total Volume25  μlThe customized primer sequence is below:  AB1Target
  GeneSequence2SARS CoV-2 nucleocapsid (N)
  gene; 2019-nCoV_N1- Forward PrimerGAC CCC AAA ATC AGC GAA
  AT3SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N1- Reverse PrimerTCT GGT TAC TGC CAG TTG
  AAT CTG4SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N2- Forward PrimerTTA CAA ACA TTG GCC GCA
  AA5SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N1- Reverse PrimerGCG CGA CAT TCC GAA GAA6SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N3- Forward PrimerGGGAGCCTTGAA TAC ACC AAA
  A7SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N1- Reverse PrimerTGTAGCACG ATTGCAGCATTG8RdRp gene
  nCoV_IP2 Forward primerATGAGCTTAGTCCTGTTG9RdRp gene
  nCoV_IP2 Reverse primerCTCCCTTTGTTGTGTTGT10(Internal Control) RNAse P
  Forward PrimerAGA TTT GGA CCT GCG AGC G11(Internal Control) RNAse P
  Reverse PrimerGAG CGG CTG TCT CCA CAA
  GT
10 µl
AB1SuperScript® III RT/Platinum® Taq Mix0.5 μl22X SYBR® Green Reaction Mix12.5 μl3Forward Primer0.125 μl4Reverse primer0.125 μl5Nuclease free water1.75 μl6Extracted RNA10  μl7Total Volume25  μl
PCR Slots 1,2,3 have primers targeting the N1, N2, and N3 genes respectively. PCR Slot 4 has a primer targeting the RNAse P gene (Internal control).
AB1Target
  GeneSequence2SARS CoV-2 nucleocapsid (N)
  gene; 2019-nCoV_N1- Forward PrimerGAC CCC AAA ATC AGC GAA
  AT3SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N1- Reverse PrimerTCT GGT TAC TGC CAG TTG
  AAT CTG4SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N2- Forward PrimerTTA CAA ACA TTG GCC GCA
  AA5SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N1- Reverse PrimerGCG CGA CAT TCC GAA GAA6SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N3- Forward PrimerGGGAGCCTTGAA TAC ACC AAA
  A7SARS CoV-2
  nucleocapsid (N) gene; 2019-nCoV_N1- Reverse PrimerTGTAGCACG ATTGCAGCATTG8RdRp gene
  nCoV_IP2 Forward primerATGAGCTTAGTCCTGTTG9RdRp gene
  nCoV_IP2 Reverse primerCTCCCTTTGTTGTGTTGT10(Internal Control) RNAse P
  Forward PrimerAGA TTT GGA CCT GCG AGC G11(Internal Control) RNAse P
  Reverse PrimerGAG CGG CTG TCT CCA CAA
  GT
?. [Reverse transcription PCR]
PCR setup for 40 cycles   ABC1cDNA synthesis50°C3 minutes2Initial denaturation95°C 2 minutes`3Amplification (40 cycles)95°C 15 seconds460°C30 seconds
ABC1cDNA synthesis50°C3 minutes2Initial denaturation95°C 2 minutes`3Amplification (40 cycles)95°C 15 seconds460°C30 seconds
?. [Reverse transcription PCR]
After completion of the PCR cycle, results are noted by determining the increase in the fluorescence intensity in the PCR slots.
The results are interpreted invalid if:1) If the RNAse P (Internal control) shows no increase in fluorescence intensity (-).2) If the positive control (P) in the test cartridge shows no increase in fluorescence intensity (-).2) If the negative control (N) in the test cartridge shows an increase in fluorescence intensity (+).
1)The result is interpreted as positive if: Any of the PCR slots 1, 2, and 3 exhibit an increase in fluorescence intensity (+).2)The result is interpreted as negative if: All the PCR slots 1, 2, and 3 show no increase in fluorescence intensity (-). In case (1) and (2), the positive control and RNAse P (Internal control) show an increase in fluorescence intensity (+), and the negative control shows no increase in fluorescence intensity (-).