Molecular Diagnostics - Science topic

For diagnostics of Avian infectious bronchitis viruses, I use an end-point RT PCR reaction, and before the RT srage I do a primer annealing stage with the target reverse primer (2uM). My queation is: If I have 2 targets, is it right to use a 1:1 mixtue of their reverse primers (2uM) instead of using rendom hexamers, in order to get better sensitivity for these two targets amplified from the same RT product?

We performed PCR with blood samples and blood culture bottles to correlate the significance of the sample type.

As a validation run, we used samples from K2-EDTA containers and blood culture bottles for a multiplex PCR run. The results show the expected results in K2-EDTA sample and there was no detection in the blood culture bottle sample, even Internal Control (IC) was not detected.

Please anyone expert in this field, suggest the reason for this failure and how we can overcome this issue.

I really appreciate any help you can provide.

Can someone please explain to me what is

thermoplastic microfluidic and how it can be useful in molecular diagnostic labs?

Thanks in advance.

We have a Qiagen QIAquant PCR instrument at COVID 19 Molecular Diagnostic Lab and we have used standard 8-well strips 0.2 ml so far. Now we are going to utilize standard 96-well microplate. Is there anybody worked with these microplates? Can we reuse the plates? If yes, after finishing a run, How should we clean up the plate and prepare for the next run? and How many times can we use them? Finally, I should mention our work is so sensitive because we do diagnostic tests.

Thank you so much

Does Autoclaving and gamma irradiation remove DNA enough so that no DNA fragment will be amplified in PCR?

Molecular methods are widespread in modern zoology. And I'm not sure if they are universal enough to be applied in all scientific fields.

Are examples of failure of molecular diagnostic systems for identification in your work?

Dear colleagues, we (with my collaborators) develop an interesting multi-wallet carbon based nanosensors for for amplification-free DNA detection. According to literature this kind of sensors is non very popular but there are enought evidence of their potential in molecular diagnostics. May be anybody use (used) nanosensor in their work? Could you give me feedback? We would like to improve and adopt our sensors for clinical diagnostics.

Now we able to recognize SNP's and specific viral/bactrerial NA in samples after routine column-based NA purification. We do not use any amplification. The detection is non-fluorescent.

I am interested to explore the Genetics of Complex Human Diseases (infectious diseases and cancer). Aiming to evaluate the transcriptional and proteomics regulation of genes/ proteins involved in disease progression and proliferation, Molecular insight of Host-pathogens interaction, genetic variation and drug-resistance mechanisms, molecular diagnostic, prognostic and predictive Biomarker of the diseases.

Being an early career researcher I have submitted several projects for funding based on molecular biology but no successful results yet. I am still trying very hard to get funds and collaborate with national and international researcher.

I have an expertise at OMICs level (DNA/RNA/Proteins techniques), if you have any related interest, you are welcomed for collaboration, we can share the present resources or can wright a joint research projects, submit somewhere for funding....

Hope to work together for the betterment of our coming generation...

Best Regards

Studying the sensitivity of methods used in HLA typing

We are on the path of developing Genotype panels for QC evaluation of molecular diagnostic kits (HIV, HBV, HCV). What would be a better method to approach - Sanger sequencing method or NGS platform. The sample size is tentatively 1000 numbers.

I have developed RT PCR mastermix , now its required to add stabilizer in it? if yes which stabilizer we can use at which concentration.

From the methods I have come across, I haven't seen any that claim to be capable of purifying nucleic acid from bacterial, fungal and viral cells. Is there a commercially available method out there capable of this? Also, most methods seem to focus on blood. Are there any methods out there for sputum? Thanks for your help.

Molecular diagnostic of BPH Nilaparvata lugens (Stal) biotypes.

A couple seeking premarital genetic counseling:

Male: heterozygous for ∆3.7 single gene deletion mutation (alpha thalassemia trait)

Female: heterozygous for IVS II-1 G>A mutation (beta thalassemia trait)

What would be the phenotype of a kid carrying both traits?

Thank you in advance.

molecular diagnosis

My laboratory is trying to develop a qPCR assay to detect rodent fur mites Radfordia affinis and Radfordia ensifera. However, there are no published nucleotide sequences in NCBI for either species, nor publications with primer/probe sequences in Pubmed.

Thank you for your help in advance.

We are about to start our diagnostic lab for Fragile X, and the real-time machine that appeals to us is LineGene 9600 Plus (https://www.biosan.lv/en/products/molecular-diagnostics/amplificationpcr/linegene-9600-plus-real-time-pcr-detection-system)

Its specifications are satisfactory. However, we could not find any feedback.

So if any of you has worked with this model, please, let us know if it is worth purchasing it. What are pros and cons that you have encountered while working with this qPCR machine. 

Your feedback may turn out to be decisive for us.

Lela

I had already quantified my DNA using a Spectrophotometer, and most of the samples were around the 200-300 ng/ul. When I quantify using Picogreen assay, my standards range from 10-1000 ng/ml. I know the conversion is 1000ng/ml= 1ng/ul... So, if one of my samples concentration with the picogreen is 600ng/ml that means I only have .6ng/ul?? 

I have been trying to find out about this, maybe I'm not doing the conversion right? Please, someone help. 

i want to know the genetic disease in india for which molecular diagnostics is used the most

I want to avoid chaotropic reagents as they are supposedly toxic and as I am developing a point of care diagnostic protocol for ZIKA to be used on the field (not under normal laboratory conditions), I would prefer a non-toxic reagent.  

Can anyone tell me what type of chemical reactions happens in diagnostic kits like- pregnancy detection kits? From where I can get details about similar kits used in diagnostics?

I want to have list of such ready to use reliable diagnostic kits. Whomever knows any just reply in answer. Thanks.

I have to be prepared if the virus comes up in Europe or Germany.

Hi

I would like to know why beta globin is used in real time PCR for HPV detection?

During specificity test of a diagnostic, when it challenged against plasmid DNA from a non-target species at a very high concentrations of DNA non-target (10E8 copies/ul) there was amplification. However, at lower concentrations this did not occur. Non-specific amplification may have occurred due to stochastic events, ie, since all the components necessary for amplification to occur occur (except for the target DNA), the physical interaction between the molecules could have favored non-specific amplification, considering this extreme situation of High concentration of non-target DNA?

Idea about universal primer of low risk Human Papilloma Virus? Only  universal low risk Primers?

Can Cas9/gRNA recognize SNP? What is the sensitivity of Cas9/gRNA? If the sensitivity is not good, is there any enzyme in Cas family can recognize SNP? or have higher sensitivity? Thanks!

The delivering time of the dig-labeling kit(Roche) is so long. Is there any other effective ways to label the RNA fragments with digoxin ?

I need to detect and quantify simultaneously 6 different bacterial gene from stool sample. Is it possible by using Applied Biosystems™ TaqMan® QSY ™ probe to optimise the required qPCR in BioRad CFX96 system.

Is there any research/groups working into miniaturizing AC Pulsed Field Gel Electrophoresis? Like if you want to use it a lab-on-a-chip or similar formats.

How effective and reliable would "Ion AmpliSeq™ Cancer Hotspot Panel v2" be for identifying variants of oncogenes and tumor suppressors genes in xenograft tumor? 

Expecting comments from users of this panel. 

I made an in-vitro transcription of a nuceoprotein gene (cloned in pGEM-T) to determine the viral load in infected patients with RT-qPCR assay. After obtaining the apropriate quality and quantity of RNA, the calibration curve was obtained with serial dilutions of the transcript.

When I made the calibration curve, those dilutions more than -8 (with a Ct=30) resulted negative in the RT-qPCR. However, there are a few samples of infected patients that are detected by this RT-qPCR with Ct values more than 30 (eg. 33, 36). Therefore, I cannot determine the viral loads of those samples considering that my calibration curve doesn't include Ct values more than 30.

Do you have any idea why this happens and how can I quantify these problematic samples?

Anyone know what is the minimum concentration (copies/uL) that should obtained?

Hey,

In order to standardize and optimize positive controls for Real-time PCR in our laboratory, and also to gain time, we were wondering the stability of plasmid dilutions for semi to long term use in our diagnosis routine.

We want to know if we can store our positive controls dilutions at 4°C for a month without loosing any stability ?

I'll appreciate any knowledge or experience or feedback on that subject

cheers.

I am planning to perform Methylight to bisulfite-converted cell-free DNA isolated from blood(Serum/plasma). Can you please suggest which DNA Polymerase with 5' to 3' exonuclease activity works best with cell-free DNA? (I will use Stratagene Mx3005p Real-Time PCR system)

Can anyone give me the exact THP protocol for DNA extraction. I have been already using this based on the following reference, but unable to optimize it yet.

So the DNA is extracted then converted via bisulphite treatment before the PCR.

Wondering is there a good method for optimisation of the PCR before i move on to clinical samples.

Thinking spiking known pos cells in a negative background or spiking converted DNA into a non converted DNA background

Thanks

What are the methods to reduce PCR bias. which is the technique having the least PCR bias?

HCV genotyping using conventional methods

Hi 

If I have 200-1000 SNPs I need to test on regular basis with clinically validated way ( direct-to customer service) which way I may choose e.g. custom made Microarray or Taqman probes ( I need the cost as least as possible not exceed 100$ for 200 SNPs if possible).

-DNA from Urine

- Toxoplasma Gondii

- LAMP

currently PVL is detect by PCR only, and its fatal toxin which need early detection specially among inpatients and those in ICUs

Dear friends

I am trying to construct few deletion mutants of Mycobacterium abscessus using the pJV53 recombineering system. I have transformed the M. abscessus cells harboring the pJV53 plasmid (carries recombinase, Km resistant) with the linear PCR product which contains the flanking regions of the gene to be deleted and the zeocin cassette. I selected the colonies in 7H10 plates containing kanamycin and zeocin. However, when I do PCR to confirm the incorporation of zeocin cassette into the gene to be deleted, I get the same band as that of wild-type. I checked the resistance of M. abscessus WT cells to kanamycin and zeocin and the cells didn’t grow in both the antibiotics.

Any help on this would be greatly appreciated.

Warm regards

Bindu

Does someone knows a good book or a PDF file on the subject of DNA repair? Especially with BRCA1 as a key gene? It is for a thesis defence. So it shouldn't be too extensive. 

In search of an adequate prognostic model for early recognition of delrium in Cardiothorasic ICU patients

I performed Mycobacterium marinum genomic DNA (mmar_2193) using specific primer (FP-ttcatatggattttgatgcgctg and RP 1-ttaagcttctagttttgccgccgcgc and RP 2-ttaagcttgttttgccgccgcgc) 

PCR master mix

5x Reaction buffer- 5ul

GC enhancer        - 5ul

dNTP                     - 1ul

DNA                       - 1ul

FP                          -1ul

RP                         - 1ul

miliQ water              -31.5

q5 polymerase       -.5 ul

total                        - 50 ul

98⁰c   98⁰c  64⁰c*  72⁰c     72⁰c      4⁰c

 2min   30s  45s    30s    10min        ~

1cycle           30cycle           1 cycle

i use gradient angling temperature 63-65 °C.

but all time comes nonspecific band. My band size 636bp.

what did i do wrong?

please suggest to me! 

I did COLD-PCR for JAK2 wildtype and mutant samples. The previous results were okay, the peaks were around 76-77 celcius. Now the peaks are around 77-78, I wonder what happened. My samples are kept in -80 celcius freezer, I have tapped and spinned down them and my master mix before running them on RotorGene.

Hi, I ran two gels couple of days ago and did not get good bands. I initially thought that it was my secondary that's not working well. But when I checked the gels I realized that there were thick bands at the low molecular weight marker levels.

Every time I do transfer I would check if the ladder is transferred onto the blot (that's how I determine if the transfer is successful). The ladder was on the blot but why are the proteins not transferring??

Is there a particular feature or a direct connection that can explain why immunohistochemical expression of mutant protein p53 is increased in obese and diabetics (type 2 diabetic) patients at diagnosis of colorectal adenocarcinoma compared to non -obese diabetics versus non-diabetics obese/non obese?

Study included immunohistochemical analysis of  2 proteins Bcl2 and p53 (both monoexpression and coexpression) in 100 newly  diagnosed colorectal adenocarcinoma specimens divided  equal in 2 groups diabetics (type 2 diabetes mellitus already existing at diagnosis of colorectal cancer)  and non-diabetics .The preliminary statistics results have not indicated a significant difference between the 2 groups, in terms of Bcl2, but  indicates an increased expression of p53 only in the  diabetic-obese group compared to  non-diabetics obese or non-obese (statistically significant ) which theoretically indicates a contact point between obesity, cancer and diabetes. Interestingly moderately low degree of differentiation is associated with both diabetes and the presence of p53 positivity; G2-p53 associations is an intriguing part. Bcl2 expression was low and insignificant in both group.

How to estimate the hpv antigen content by ELISA test ?

What type of elisa kits are using for estimation of hpv antigen content and equipment .

I am having problems getting any detected insulin from dried blood spots after extraction with PBS+Tween.

I am using a common biochemical analyzer (electroluminescence) instead of ELISA (which could be the problem since ELISA detection has been successful according to bibliography).

Any ideas?

I am working on  Molecular diagnostics of Human Papilloma Virus through Conventional PCR, but there is no Internal Control on the Kit which I am using for it so I just want to use human beta globin gene primer with the master mix of that kit but i don't have any idea regarding this. Can anyone help me out of this......... 

We have diagnosed a girl with McKusick-Kaufman syndrome (MKS) based on clinical presentation with hydrometrocolpos, polydactyly, and congenital heart disease. We want to confirm the diagnosis at the molecular level, but we do not know where to do this.

Has anyone suggestions about laboratories to perform mutational analysis of MKKS gene? 

Usually we calculate the force constants for bond length, bond angle and dihedral angle. I want to calculate the force constant for entire molecule.

I want to quantify Fe, Cu, Ni, Sn and Cd from urine. What are the procedures for posterior analysis of the sample in ICPMS? Should I acidify the urine?

2

i want to start molecular diagnosis unit in my lab. so i need highly specific kits for different genetic disease. I don't have Real-Time PCR, I just have conventional gradient PCR machine, so i need that kits only which can work with it.  For Qualitative analysis ( Diagnosis) only.

- sample 9 has a negative result using exactly the same mastermix I used for the negative control. 

- used a 100 bp molecular weight ladder

- negative control: water

- 9-14 are the samples 

Hi

I am doing cDNA subtractive hybridization to know the differential expressed genes using Clonetech Kit.  First, I did PCR using random primer, but did not get any amplification and then I did PCR utlizing nested primer for 3 different hybridized sample.  The 1st and 2nd samples are diluted where as the 3rd is not diluted.  I have used Emerald Master Mix for PCR and loaded 5 microliter.  I have attached the gel picture, please give your valuable suggestion.  Thanks

miRNA is proposed to be clinical biomarker in many diseases, as well acute and chronic ones. I think that there is a need to validate extraction protocols to introduce miRNA into molecular diagnostics as a valuable parameter .

There are many commercial kits and in house methods you can recommend to extract miRNA from body fluids, but it there is always a problem to obtain good concentrations of miRNA to perform high-throughput methods or even e sequencing. There are some protocols published but I want to activate people who REALLY deal with clinical samples, and can share their authentic experience.

We tried several times to isolate RNA from HK-2 cells with the qiagen RNeasy kit (with RLT buffer). We can do the isolation of RNA (with Qiagen kit) from other lines of human immortalized proximal tubular epithelial cells without any problem, but from the HK-2 cells it's very hard to get RNA. We already tried to homogenize the sample better, using needle and syringe. We also already added the RLT buffer immediately to the culture dish (instead of to the pellet) and scraped the cells. All without improvement...

What else could we try?

Please suggest steps for detecting amplification by loop mediated isothermal amplification (LAMP) assay using lateral flow dipstick (LFD). The FIP is biotin labelled and probe is FITC labelled since i am using LFD from Milenia.

I was able to get very faint test line by incubation of probe (Tm 67) with amplicon at 65 degree celcius for 5 minutes. But the result is non-reproducible.

I have tried various concentration of probe and different hybridization temperature based on published literature. I have tried with fresh reagents also. There is no signal on test line despite very high amplification.

Please suggest how to troubleshoot the problem or how to carry out LAMP assay with LFD.

What will be the consequences out of this?

Circulating tumor DNA (ctDNA) is now widely investigated as a biomarker in translational and clinical research. However, despite the growing field of clinical applications, the biology of ctDNA remains unclear. In trying to learn about the origins of ctDNA, nature provides us with very few clues. One of the important accessible parameters is the size of those DNA fragments. In addition, a well-informed model of these sizes and biases can help design more efficient and accurate diagnostic methods. In PNAS, Jiang et al. take an important step in that direction.

We used massively parallel sequencing to study the size profiles of plasma DNA samples at single-base resolution and in a genome-wide manner. We used chromosome arm-level z-score analysis (CAZA) to identify tumor-derived plasma DNA for studying their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These findings have shed light on fundamental biological characteristics of plasma DNA and related diagnostic applications for cancer.

The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-level z-score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.

Jiang P et al.: Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci U S A. 2015 Feb 2. pii: 201500076.

I am new to the field of circulating nucleic acid detection. I would like to search for microRNA down/upregulation in blood samples from patients suffering from certain type of cancer. I have never tried to isolate and quantify extracellular micro-RNA. Have you tried it? Maybe there is a good kit you can recommend? Is the 30-5-5 min serum centrifugation necessary?

One of my collegues accidently put the silica columns out of the refrigerator about six months ago. I have known that the binding efficacy will be dropped in this situation but, is there any one who experienced similar situation like this?

I have dissected out mouse hippocampus and want to homogenize it for analysis of phosphorylated and aggregated tau. We have dounce homogenizers but they feel too big for such small tissues. We also have a TissueLyser from Qiagen (normally used for RNA-prep) which uses a bead to disrupt the tissue, but I have been told that too strong mechanical disruption can destroy the large aggregates. Are there any do's and don't-s for this type of sample preparation? 

I extracted whole blood samples using the Qiagen DNeasy blood and tissue kit. When quantifying the DNA on the Qubit, I get baffling low DNA concentrations. Is there something that I might be doing wrong? Is there a way to increase my DNA yield after extractions?

Thank you!

We have since 2 Month the QX200 droplet digital PCR-equippement from BioRad. We want to introduce this as soon as possible into Routine diagnostics. as it is a new technique only Little experience can be shared. I would like to share mine. Therefore I ask here to get in contact with me to share experience in the field of Food diagnostic.

René

I am detecting HPV mRNA by real time PCR. the PCR is of 50 cycles. There are many samples which show the Ct value as high as 40. Does it mean that they contain the HPV mRNA? should they be considered as a positive sample? where should I draw a line to consider them as positive or negative? 

I want to amplify the uncharacterized gene. First step, I will aplify and sequencing full cDNA of gene.

What is the best (quality and price) the RACE kit ?

(First choice, invitrogen  and   SMARTER RACE, takara  or etc.)

thank you

Is there an agent for virus inactivation that can be used in whole blood samples, that still allows to make routine tests in those samples, like blood cell count and blood chemistry?

What can be done to improve the sensitivity and specificity of the tests, aside from using proper controls?

What primers are best for Toxoplasma detection?

Real-time PCR?

Analyse cDNA to confirm the presence of living Toxoplasma - is this a viable idea?

Is the detection of antibodies considered mandatory to prove Toxoplasma infection, or will PCR results suffice if done properly? Or are there some problems that I don't know about?

Will be most grateful for any ideas on the topic.

Immunohistochemistry is a method for demonstrating the presence and location of proteins in tissue sections. Though, less sensitive quantitatively than immunoassays such as Western blotting or enzyme linked immune sorbent assay (ELISA), it allows the study of the distribution and localization of specific cellular components by providing supplemental information to the routine morphological assessment of tissues. This is especially useful for assessing the cellular markers that define specific phenotypes then providing important diagnostic, prognostic, and predictive information relative to disease status and biology. In general, the information gained from IHC combined with microscopy literally provides a “big picture” that can help make sense of data obtained using other methods.

If that is possible would 2-step method more useful than 1-step?

PD is a potential by-product in PCR, a PD consists of primer molecules that have attached (hybridized) to each other, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.

I am working in a project that l use both one step and two steps methods, but l would like to know which of them more useful and specific.

Genomic imprinting differs from that of ordinary Mendelian rules of inheritance, as in the first aspect the gene is expressed by one allele from one parent and the other is silenced by imprinting; where in Mendelian the gene is expressed by two alleles coming from both parents. The imprinted genes are maintained in somatic cells, where in germline cells it is erased and re-established during development.

With the growing number of biomarkers regularly discovered, going through clinical trials and more slowly translating into the clinic, what options are available to shortlist and find biomarkers? For example for:

- Developing new diagnostics/drugs

- Researchers seeking to make new biomarker discoveries 

- Selecting biomarkers for disease diagnosis/treatment 

- other ways biomarkers are used/needed for projects

I am going to generate antibodies by injecting a mixture of Freund's adjuvant and purified protein into mice. Should I use intraperitoneal injection or subcutaneous injection?

I am interested in the microparticles' functions in blood. I want to determine the levels of some microRNAs included microparticles by real-time qPCR. As the level of microparticles in blood is limited, I want to know if the level of the included microRNA is sufficient to be detected?