Thank you so much for sharing sir. You're such an absolute great teacher. Now i have a perfect understanding of this aspect. I am very grateful.....Big thumbs up
Thank you so much for your comments... And i am happy you understand it....if any problem or queries then text a message i will explain it...will do it in English ONlY
Nice explanation sir, how we can set up an appropriate threshold line if the kit is not recommending the automatic threshold? Are there any criteria for the same? How you have ruled out IC amplification failure is due to improper sampling not due to PCR inhibitors like ethanol carryover? Is it mandatory to keep an extraction control for every run sir?
1- for threshold line, no RTPCR kit recommend about the threshold line settings...it's all about instruments... Positive control criteria is amplification with valid CT... But no demarcation with respect to line in kit manuals. 2- IC amplification due to PCR inhibitors can be possible, but it's practically impossible to find out during Pandemic state... And this comes due to experience of testing more than 8 lakhs samples in last year... You have to set your standards, because when we are testing daily 5-10k samples per day... Then we have to achieve the target with some fixed algorithm while maintaining the quality standards. 3- Ideally yes, because it reflects that your RNA team and the reagents of RNA are performing well....in case any time... PCR fails... Hope this is useful, please subscribe the channel 🙏...and forward it as much as possible
Sir my qus is : 1. What is ORF gene : is this denote a perticular faimly of the virus? eg. ORF will be same for all the SARS faimly members. Is it? 2. What in N gene or E gene? 3. What exogenous & endogenous gene or IC? 4. If ther is any kind of noise in the graph, how to correct it, is there any slop corrections or outlier removal option like we have in Rotor gene QIAGEN RT PCR software.
1-ORF gene is specific for this novel corona virus 2019 2-represents for envelope protein and that is non specific to novel guruna virus 2019 this is common for SAARC because this is an envelope protein so that is why on the basis of only gene positive you cannot give the patient as noble Quran virus 2019 positive 3-exogenous and endogenous control or internal controls are the controls of the RT-PCR, That very according to the kit which we used and if internal controls are not properly amplified as per manufacturers instruction then you cannot clear the batch there is some problem in execution of the PCR 4- noise in any graph can be rectified, all depends on the graph images, but it must be sigmoid.... threshold can be changed but it's all depends
These all standards are well defined and has been mentioned in the leaflet....and only two will be amplified....if ROX is not amplified then all controls works well and test valid as per manufacturers instructions
Thank you very much sir, it's was very informative. As I have very basic knowledge about pcr. Sir I had doubt about how to set the threshold line and baseline
Thank you for asking related query, Just remember one thing, baseline or threshold line play a very important role in declaring the results. Only scientific personal can understand this, First of all, if you have CFX then it's very good.....no need to set the line, machine will automatically sets the threshold. If you are not satisfied with the line, then ONLy you can change the settings. Only in extreme cases..... Otherwise, you will set the threshold according to control, positive control from where amplification has been Started.... I will try to make a short vedio on that...which will give you confidence...
Threshold value, is very important for the one who is reporting and handling the software. This will completely change the results during analysis of someone will play or tried to adjust the threshold according to the batch controls....
So, I suggest first try to analyse first with the system generated threshold line, because after the run will be finished, the software will define the threshold on the basis of all 96 samples amplification CT
I will explain in another vedio about the truth of CT and it's importance in clinical investigation, because everyone is now confused with their own CT values....
@@TheScientificGurus full explanation of RTPCR from basics i.e. the role of master mix , primer pro, RNAwater , what they do , how did pcr do it. Full explanation of extraction procedures, role of RNA lysis etc.
@@poornimasirvaiya7949 thank you for mentioning the same... Today I already planned to post video on master mix.... Preparation with practical experience
Sir, here people are mixing everything in the mastermix vial, except the negative and positive control, and then they put 15 microlitre in each well, and then put 10 microlitre rna to make the volume of 25 microlitre. Is this a good practice, or we should put the mastermix, enzyme mix and primer probe mix one by one???
See, thank you for your query. I want to know which kit you are using. Because most of the companies are giving a single tube master mix and primer probe mix tube, means two tubes ONLy. In that case you have to mix only these tubes and go ahead with distribution. And if you are using enzyme, buffer and probes seperately I guess u r using invitrogen, then you have to make ONLy required volume according to the number of samples... Otherwise you will get irregular peaks in every well...probes will be degraded
Thank u so much Sir, for such an early reply... Your videos are just great.. I just saw once and every concept has come clear.. Thanku so much Sir.. Regards..
@@TheSuperanupamkumar Actually this kit is not satisfactory as compared with other kits. TruPCR sometimes gives confusing results, we have used TaqPath, Meril, GB Sars, more than 15 COVID kits... I will.not recommend that kit for diagnosis.
As per kit both the primers are selected from conserved regions of ORF and N gene, what if mutation is in ORF and not getting amplified then shld I relay on N. Can u plz through some light on screening and confirmatory attribute of ORF and N.
N gene is altogether confirmatory target, and if n gene is significantly amplified then results will be positive. So you have to reply on N gene because N gene has less nucleotide variation than ORF1ab that may make the detection of the N gene more stable than that of ORF1ab. Nucleocapsid, a type of structural protein of SARS-CoV-2 is encoded by 908 nucleotides N gene at the near 3’ end and it’s a potential target for the detection of virus as recommended by CDC. So reply on N gene amplification as N gene could be detected separately for monitoring viral nucleic acids. Their are many reports that showed results with viral nucleic acid tests that can continue to be positive for up to 40 days in some patients. I hope this will clear some of your doubts, I can share some important links which help to go through the AVAILABLE literature or study..... Regards Dr. Mayank
Thank you for your suggestions, you need to know the diagnostic procedures of genes step by step or the sequencing procedure...and if sequencing the it is by two methods.. capillary based or WGS
I can understand you want to know the reporting system from laboratory to hospital, especially sufalum... Any specific section in sufalum because it's a very complex and long lecture
Make it by default from system....if possible no need to change.... And if need to be set, then place it when amplification started in positive control...
Which machine you are using???? If CFX 96, then i suggest do not disturb the baseline....by default software will provide you best fit after the completion of run....if you want to set manually then i will discuss it separately
Dear, I am Dr. Jogendra. Excellent Video having important knowledge.
Thank you so much for watching and comments.... please subscribe the channel and support 👍
Bahut badiya sir
Thanks.....any doubts just text
Great sir, excellent explain .
Thank you for watching...
ua-cam.com/video/JWGyzfLfpQI/v-deo.html
How to set a protocol....
Kindly subscribe and follow it
very helpful video. thanks bro
Always welcome
Very informative sir, i never see this type of detailed video. Thanks
Thanks dear for your comments... If you want any doubts or session on PCR then let me know
Thank you for information sir
Please subscribe and follow the channel...
Thanks alot sir. We are proud of you that you are putting so much efforts despite your busy schedule.
Nicely explained
Thanks for your comments.... It's actually the initial draft, i was actually unable to explain in detail.....
So Thankful to you sir.
It was accurate, precise and Lucid! 🙂🙏
Thanks for your comments, stay tuned and suggest anything related with molecular biology
you are a great teacher sir🙏
Thanks a ton
Thank you so much for sharing sir. You're such an absolute great teacher. Now i have a perfect understanding of this aspect. I am very grateful.....Big thumbs up
Thank you so much for your comments...
And i am happy you understand it....if any problem or queries then text a message i will explain it...will do it in English ONlY
Please subscribe the channel and forward it to everyone, support the channel...
And post what next you want
This video worth more then gold 🙂❤️
Thanks for your comments Dear....
Thanks a lot. Very informative video. Please also explain background noise and how to set threshold
Sure I will
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Very interesting, good explanation..now I understood how to interpret graphs. Can you please make similar video with using Quiagen PCR kit..
Sure... Thanks
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Can you please explain which PCR kit yoy wann know, because COVID qiagen PCR kit is not available... Qiagen which targets yoy wann know
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Visit our New RTPCR vedio
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Nice explanation sir, how we can set up an appropriate threshold line if the kit is not recommending the automatic threshold? Are there any criteria for the same? How you have ruled out IC amplification failure is due to improper sampling not due to PCR inhibitors like ethanol carryover? Is it mandatory to keep an extraction control for every run sir?
Very nice questions....
I will address one by one...but it requires a lot of explanation.....
1- for threshold line, no RTPCR kit recommend about the threshold line settings...it's all about instruments... Positive control criteria is amplification with valid CT... But no demarcation with respect to line in kit manuals.
2- IC amplification due to PCR inhibitors can be possible, but it's practically impossible to find out during Pandemic state... And this comes due to experience of testing more than 8 lakhs samples in last year...
You have to set your standards, because when we are testing daily 5-10k samples per day... Then we have to achieve the target with some fixed algorithm while maintaining the quality standards.
3- Ideally yes, because it reflects that your RNA team and the reagents of RNA are performing well....in case any time... PCR fails...
Hope this is useful, please subscribe the channel 🙏...and forward it as much as possible
Visit our New RTPCR vedio
ua-cam.com/video/EyiobQXjnVk/v-deo.html
if possible kindly make a practical video on NGS on Ion chef torrent platform by thermo fischer
The only one superclear, congratulations!
Thanks any queries related to PCR or covid19 or any biology methods...are always welcome
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Visit our New RTPCR vedio
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Thanks alot for this clear explanation 👍 👌
Thank you so much, please subscribe and comments for any molecular diagnostics related topic..
I m going to upload very soon about the controls used in the PCR such as PC, NC and EC
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Sir my qus is :
1. What is ORF gene : is this denote a perticular faimly of the virus? eg. ORF will be same for all the SARS faimly members. Is it?
2. What in N gene or E gene?
3. What exogenous & endogenous gene or IC?
4. If ther is any kind of noise in the graph, how to correct it, is there any slop corrections or outlier removal option like we have in Rotor gene QIAGEN RT PCR software.
1-ORF gene is specific for this novel corona virus 2019
2-represents for envelope protein and that is non specific to novel guruna virus 2019 this is common for SAARC because this is an envelope protein so that is why on the basis of only gene positive you cannot give the patient as noble Quran virus 2019 positive
3-exogenous and endogenous control or internal controls are the controls of the RT-PCR,
That very according to the kit which we used and if internal controls are not properly amplified as per manufacturers instruction then you cannot clear the batch there is some problem in execution of the PCR
4- noise in any graph can be rectified, all depends on the graph images, but it must be sigmoid.... threshold can be changed but it's all depends
Any other doubt related to PCR you can ask me anytime
Thank you so much sir !!
Please visit our new video on RTPCR master mix
ua-cam.com/video/Gs6Z5ur75xw/v-deo.html
But how about positive FAM, positive HEX, Negative ROX?
These all standards are well defined and has been mentioned in the leaflet....and only two will be amplified....if ROX is not amplified then all controls works well and test valid as per manufacturers instructions
Thank you very much sir, it's was very informative. As I have very basic knowledge about pcr. Sir I had doubt about how to set the threshold line and baseline
Thank you for asking related query,
Just remember one thing, baseline or threshold line play a very important role in declaring the results.
Only scientific personal can understand this,
First of all, if you have CFX then it's very good.....no need to set the line, machine will automatically sets the threshold. If you are not satisfied with the line, then ONLy you can change the settings.
Only in extreme cases.....
Otherwise, you will set the threshold according to control, positive control from where amplification has been Started....
I will try to make a short vedio on that...which will give you confidence...
Please subscribe to the channel 🙏🙏🙏
Visit our New RTPCR vedio
ua-cam.com/video/EyiobQXjnVk/v-deo.html
Please also explain noise and how to set threshold values ??
Threshold value, is very important for the one who is reporting and handling the software. This will completely change the results during analysis of someone will play or tried to adjust the threshold according to the batch controls....
So, I suggest first try to analyse first with the system generated threshold line, because after the run will be finished, the software will define the threshold on the basis of all 96 samples amplification CT
I will explain in another vedio about the truth of CT and it's importance in clinical investigation, because everyone is now confused with their own CT values....
Please visit our new video on RTPCR master mix
ua-cam.com/video/Gs6Z5ur75xw/v-deo.html
Visit our New RTPCR vedio
ua-cam.com/video/EyiobQXjnVk/v-deo.html
Thank you so much sir 🙏🙏 very helpful. Clear explanation. 🙏🙏
Thank you for your comments please subscribe the channel for ONLy Scientific updates 🙏🙏🙏🙏
@@TheScientificGurus subscribed 👍
@@poornimasirvaiya7949 any biomedical applications or RTPCR related topic of required for discussion... please drop a message....
@@TheScientificGurus full explanation of RTPCR from basics i.e. the role of master mix , primer pro, RNAwater , what they do , how did pcr do it. Full explanation of extraction procedures, role of RNA lysis etc.
@@poornimasirvaiya7949 thank you for mentioning the same...
Today I already planned to post video on master mix.... Preparation with practical experience
Sir, here people are mixing everything in the mastermix vial, except the negative and positive control, and then they put 15 microlitre in each well, and then put 10 microlitre rna to make the volume of 25 microlitre. Is this a good practice, or we should put the mastermix, enzyme mix and primer probe mix one by one???
See, thank you for your query. I want to know which kit you are using.
Because most of the companies are giving a single tube master mix and primer probe mix tube, means two tubes ONLy. In that case you have to mix only these tubes and go ahead with distribution.
And if you are using enzyme, buffer and probes seperately I guess u r using invitrogen, then you have to make ONLy required volume according to the number of samples...
Otherwise you will get irregular peaks in every well...probes will be degraded
Sir, we are using truepcr SARS cov-2 RT q pcr kit (V 3.2)
Thank u so much Sir, for such an early reply... Your videos are just great.. I just saw once and every concept has come clear.. Thanku so much Sir.. Regards..
@@TheSuperanupamkumar Actually this kit is not satisfactory as compared with other kits.
TruPCR sometimes gives confusing results, we have used TaqPath, Meril, GB Sars, more than 15 COVID kits...
I will.not recommend that kit for diagnosis.
Visit our New RTPCR vedio
ua-cam.com/video/EyiobQXjnVk/v-deo.html
As per kit both the primers are selected from conserved regions of ORF and N gene, what if mutation is in ORF and not getting amplified then shld I relay on N. Can u plz through some light on screening and confirmatory attribute of ORF and N.
N gene is altogether confirmatory target, and if n gene is significantly amplified then results will be positive. So you have to reply on N gene because N gene has less nucleotide variation than ORF1ab that may make the detection of the N gene more stable than that of ORF1ab.
Nucleocapsid, a type of structural protein of SARS-CoV-2 is encoded by 908 nucleotides N gene at the near 3’ end and it’s a potential target for the detection of virus as recommended by CDC. So reply on N gene amplification as N gene could be detected separately for monitoring viral nucleic acids.
Their are many reports that showed results with viral nucleic acid tests that can continue to be positive for up to 40 days in some patients.
I hope this will clear some of your doubts, I can share some important links which help to go through the AVAILABLE literature or study.....
Regards
Dr. Mayank
Please visit our new video on RTPCR master mix.
ua-cam.com/video/Gs6Z5ur75xw/v-deo.html
Please subscribe the channel for ONLy Scientific updates 🙏🙏🙏🙏
Visit our New RTPCR vedio
ua-cam.com/video/EyiobQXjnVk/v-deo.html
Sir kindly upload the video of step by step gene sequencing process it will be very helpful for us
Thank you for your suggestions, you need to know the diagnostic procedures of genes step by step or the sequencing procedure...and if sequencing the it is by two methods.. capillary based or WGS
@@TheScientificGurus sir i want to know step by step process of how sequencing is being done ,practical approach
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Thank you so much.. 👍
Most welcome 😊
Hello @pratibha
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Sir one video on pipetting the mastermix and enzyme mix and that stuff, please..
Surely, I will update it a day or two.....
Go through this link also...
ua-cam.com/video/lSSsaz7-m4w/v-deo.html
Experiment controls in PCR
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Sir can you make a video on hospital reporting system sufalam???
I can understand you want to know the reporting system from laboratory to hospital, especially sufalum... Any specific section in sufalum because it's a very complex and long lecture
@@TheScientificGurus actually after exporting reports to sufalam how to send reports that I want to learn.
Which version your facility is using
@@TheScientificGurus latest one sir
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How to set thresold line before analysis the report
Make it by default from system....if possible no need to change....
And if need to be set, then place it when amplification started in positive control...
Visit our New RTPCR vedio
ua-cam.com/video/EyiobQXjnVk/v-deo.html
Plzzz sir show me how to set the baseline
Which machine you are using????
If CFX 96, then i suggest do not disturb the baseline....by default software will provide you best fit after the completion of run....if you want to set manually then i will discuss it separately