Sequence mismatch in PCR probes may mask the COVID-19 detection in Nepal.

•Most of the COVID-19 cases in Nepal are in the Southern districts of Nepal bordering India with travel histories to India.•Very few positive cases of COVID-19 are detected in Nepal which could either be due to early national lockdown.•Low PCR positivity rates could also be due to inefficiency of the PCR methods.•Whole genomes of 93 clinical samples from COVID-19 patients were analyzed to find the primer and probe binding sites.•Mutations in probe binding sites were found which could impact PCR efficiency resulting in false negative results.

Assessment of ERBB2/HER2 Status in HER2-Equivocal Breast Cancers by FISH and 2013/2014 ASCO-CAP Guidelines.

Importance: The 2013/2014 American Society of Clinical Oncology and College of American Pathologists (ASCO-CAP) guidelines for HER2 testing by fluorescence in situ hybridization (FISH) designated an "equivocal" category (average HER2 copies per tumor cell ≥4-6 with HER2/CEP17 ratio <2.0) to be resolved as negative or positive by assessments with alternative control probes. Approximately 4% to 12% of all invasive breast cancers are characterized as HER2-equivocal based on FISH. Objective: To evaluate the following hypotheses: (1) genetic loci used as alternative controls are heterozygously deleted in a substantial proportion of breast cancers; (2) use of these loci for assessment of HER2 by FISH leads to false-positive assessments; and (3) these HER2 false-positive breast cancer patients have outcomes that do not differ from clinical outcomes for patients with HER2-negative breast cancer. Design, Setting, and Participants: We retrospectively assessed the use of chromosome 17 p-arm and q-arm alternative control genomic sites (TP53, D17S122, SMS, RARA, TOP2A), as recommended by the 2013/2014 ASCO-CAP guidelines for HER2 testing, in patients whose data were available through Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and whose tissues were available through the Breast Cancer International Research Group clinical trials. We used data from an international cohort database of invasive breast cancers (1980 participants) and international clinical trial of adjuvant chemotherapy in invasive, node-positive breast cancer patients. Main Outcomes and Measures: The primary objectives were to (1) assess frequency of heterozygous deletions in chromosome 17 genomic sites used as FISH internal controls for evaluation of HER2 status among HER2-equivocal cancers; (2) characterize impact of using deleted sites for determination of HER2-to-internal-control-gene ratios; (3) assess HER2 protein expression in each subgroup; and (4) compare clinical outcomes for each subgroup. Results: Of the 1980 patients in METABRIC,1915 patients were fully evaluated. In addition, 100 HER2-equivocal breast cancers by FISH and 100 comparator FISH-negative breast cancers from the BCIRG-005 trial were analyzed. Heterozygous deletions, particularly in specific p-arm sites, were common in both HER2-amplified and HER2-not-amplified breast cancers. Use of alternative control probes from these regions to assess HER2 by FISH in HER2-equivocal as well as HER2-not-amplified breast cancers resulted in high rates of false-positive ratios (HER2-to-alternative control ratio ≥2.0) owing to heterozygous deletions of control p-arm genomic sites used in ratio denominators. Misclassification of HER2 status was observed not only in breast cancers with ASCO-CAP equivocal status but also in breast cancers with an average of fewer than 4.0 HER2 copies per tumor cell when using alternative control probes. Conclusions and Relevance: The indiscriminate use of alternative control probes to calculate HER2 FISH ratios in HER2-equivocal breast cancers may lead to false-positive interpretations of HER2 status resulting from unrecognized heterozygous deletions in 1 or more of these alternative control genomic sites and incorrect HER2 ratio determinations.

Picomolar sensitive and SNP-selective "Off-On" hairpin genosensor based on structure-tunable redox indicator signals.

A robust and sensitive electrochemical assay for chrononocoulometric detection of nucleic acids at a single nucleotide polymorphism (SNP) level has been developed. The assay exploits hybridization-induced conformational switching of gold-tethered TP53-specific 33-mer and truncated 20-mer hairpin DNA probes and methylene blue (MB) as an intercalating redox indicator. We show that by fine tuning of MB-DNA intercations the enhanced binding of MB to hybrids formed with a cancer-biomarker sequence can be achieved, and that results in robust "off-on" sensing of hybridization, while the stem-loop probe design allows minimized, independent of the DNA length background signals. Both DNA probes were sensitive to the presence of SNP in the targeted DNA sequence already at 10 pM. DNA levels, and the robust "off-on" discrimination of 10 pM perfectly-matched DNA from 50 nM SNP-containing DNA was achieved by time-adjusted chronocoulometry. This label-free hairpin DNA strategy allows systematic design of DNA assays for fast, robust and inexpensive genetic analysis in excessive mixtures of structurally-related DNA sequences and was used for specific analysis of prostate-cancer-realted cellular microRNA in total RNA samples isolated from LNCaP and BPH1 cells.

A gold nanoparticle based fluorescent probe for simultaneous recognition of single-stranded DNA and double-stranded DNA.

A fluorescent method is described for simultaneous recognition of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). It is based on the quenching of the fluorescence of fluorophore labeled DNA probes by gold nanoparticles (AuNPs). To demonstrate feasibility, two DNA probes labeled with spectrally different fluorophores were designed. The first DNA probe (P1) was modified with 6-carboxyfluorescein (FAM; with green fluorescence, peaking at 518 nm), while the second (P2) was modified with carboxy-X-rhodamine (ROX; with yellow fluorescence, 610 nm). The fluorescence signals of the labels are quenched if P1 or P2 are adsorbed on AuNPs. Upon addition of ssDNA and dsDNA, hybridization occurs between P1 and ssDNA to form a dsDNA. In contrast, P2 hybridizes with dsDNA such that a triplex DNA is formed. As a result, the dsDNA and the triplex DNA, respectively, are desorbed from the surface of the AuNPs so that quenching no longer can occur and strong fluorescence can be observed. Under the optimal conditions, ssDNA and dsDNA can be detected simultaneously via the green and yellow fluorescence, respectively. The detection limits can be as low as 330 pM. In particular, the method has excellent selectivity for the target DNAs over control DNAs. Graphical abstract A gold nanoparticle based fluorescent probe for simultaneous recognition of single-stranded DNA and double-stranded DNA is developed based on the fluorescence quenching of gold nanoparticles to different fluorophore labeled DNA probes.

Prototropically Allosteric Probe for Superbly Selective DNA Analysis.

Selective nucleotide recognition for biosensor evolution requires rational probe design toward the binding-pattern-susceptible readout but without serious poison in selectivity from the context sequences. In this work, we synthesized a dual-function (trihydroxyphenyl)porphyrin (POH3) to target the abasic site (AP site) in ds-DNA using the trihydroxyphenyl substituent and the tetrapyrrole macrocycle as the recognition unit (RU) and the fluorescent signal unit (SU), respectively. RU and SU are separated from each other but are prototropically allosteric. We found that an appropriate pH favors formation of the nonfluorescent quinine/pyrrole (O-NH) conformer of POH3. However, the complementary hydrogen bonding of RU in O-NH with the target cytosine opposite the AP site switches on the SU fluorescence through prototropic allostery toward the phenol/isopyrrole (OH-N) conformer, while the bases thymine, guanine, and adenine totally silence this allostery, suggesting a superb selectivity in single-nucleotide polymorphism (SNP) analysis. The role of the prototropic allostery in achieving such SNP selectivity is also evidenced using porphyrins with other hydroxyl substituent patterns. Because of the SU separation from RU, SU is not directly involved in the interaction with the AP site, and thus, the turn-on selectivity is also realized for DNA with flanking guanine, the most easily oxidized base in DNA. This tolerance to the flanking base identity has seldom been achieved in previous studies. Additionally, other DNA structures cannot bring this allostery, indicating that the combination recipe of the AP site design and the prototropically allosteric probe will find wide applications in DNA-based sensors.

Fluorescence In Situ Hybridization Probe Validation for Clinical Use.

In this chapter, we provide a systematic overview of the published guidelines and validation procedures for fluorescence in situ hybridization (FISH) probes for clinical diagnostic use. FISH probes-which are classified as molecular probes or analyte-specific reagents (ASRs)-have been extensively used in vitro for both clinical diagnosis and research. Most commercially available FISH probes in the United States are strictly regulated by the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), the Centers for Medicare & Medicaid Services (CMS) the Clinical Laboratory Improvement Amendments (CLIA), and the College of American Pathologists (CAP). Although home-brewed FISH probes-defined as probes made in-house or acquired from a source that does not supply them to other laboratories-are not regulated by these agencies, they too must undergo the same individual validation process prior to clinical use as their commercial counterparts. Validation of a FISH probe involves initial validation and ongoing verification of the test system. Initial validation includes assessment of a probe's technical specifications, establishment of its standard operational procedure (SOP), determination of its clinical sensitivity and specificity, development of its cutoff, baseline, and normal reference ranges, gathering of analytics, confirmation of its applicability to a specific research or clinical setting, testing of samples with or without the abnormalities that the probe is meant to detect, staff training, and report building. Ongoing verification of the test system involves testing additional normal and abnormal samples using the same method employed during the initial validation of the probe.

Semi-quantitative differentiation of cyathostomin larval cultures by reverse line blot.

Cyathostomins are the most prevalent horse nematodes worldwide and over 50 species are described. The eggs and the infective larvae (L3) can easily be obtained or cultured from infected horses, but cannot be differentiated morphologically at species level. A reverse line blot (RLB) method based on the hybridization of a PCR fragment with a species specific probe, has previously been developed for the differentiation of individual eggs and/or L3s, but is too labor intensive for large scale studies. In the present study a RLB method on multiple pooled L3s for the semi-quantitative differentiation of cyathostomin larval cultures was developed and validated. First, the probability of the presence of a certain species within a pool was calculated as function of the frequency and the number of L3s within a pool. Ten L3s per pool were found to be optimal. Next, the probability, the chance of occurrence was calculated when 4 pools per culture were used. The probability distributions for 0, 1, 2, 3 or 4 positive pools were transformed into the corresponding median frequency of the cumulative probability: 0.014, 0.04, 0.08, 0.16 and 0.59, respectively. Based on these calculated probabilities, RLB on 10 L3s per pool and 4 pools per sample was validated by estimating the cross-hybridization, precision and accuracy in 3 groups of horses. First, absence of cross-hybridization was confirmed by differentiation of the same L3s (160 L3s from the 4 horses from group 1) in the RLB on individual as well as on pooled L3s. Cross-hybridization was excluded for 9 of the most common cyathostomins. Next, the precision and accuracy were determined by the differentiation of 10 replicates of 3 cultures from 3 horses from group 2 (1200 L3s). The coefficient of variation (CV) was between 0 and 0.90 and the accuracy was between 0.42 and 1.73. A Monte Carlo simulation based on the observed scores and associated probability distributions gave similar results as the use of a fixed median frequency. The LPGs obtained from 276 larval culture counts from a larger cohort (23 horses, group 3) were not significantly different from the LPGs obtained from summation of the LPG per species found by RLB on pooled L3s. The RLB on pooled L3s was found therefore an useful semi-quantitative method for the differentiation of the most common cyathostomin L3, with a workload of approximately one tenth of that of the RLB on individual L3s.

ThalassoChip, an array mutation and single nucleotide polymorphism detection tool for the diagnosis of ß-thalassaemia.

BACKGROUND: The detection and diagnosis of ß-thalassaemia for populations with molecular heterogeneity, or diverse ethnic groups, has increased the need for the development of an array high-throughput diagnostic tool that can deliver large scale genetic detection. We report on the update and validation of the ThalassoChip, a ß-thalassaemia genetic diagnostic tool which is based on arrayed primer extension (APEX) technology. METHODS: ThalassoChip slides with new and redesigned probes were prepared for testing the microarray. Six hundred and sixty DNA samples collected from eight Mediterranean countries were used for standardisation, optimisation and validation of the ThalassoChip. The ß-globin gene region was amplified by PCR, the products were hybridised to the probes after fragmentation and the APEX reaction followed. RESULTS: The ThalassoChip was updated with new probes and now has the ability to detect 57 ß-globin gene mutations and three single nucleotide polymorphisms (SNPs) in a single test. The ThalassoChip as well as the PCR and APEX reactions were standardised and optimised using 500 DNA samples that were previously genotyped using conventional diagnostic techniques. Some probes were redesigned in order to improve the specificity and sensitivity of the test. Validation of the ThalassoChip performed using 160 samples analysed in blinded fashion showed no error. CONCLUSIONS: The updated version of the ThalassoChip is versatile, robust, cost-effective and easily adaptable, but most notably can provide comprehensive genetic diagnosis for ß-thalassaemia and other haemoglobinopathies.

Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar.

A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P < 0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia , Cryptosporidium sp., Escherichia coli , or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.

Confirmation of BD ProbeTec Neisseria gonorrhoeae reactive samples by Gen-Probe APTIMA assays and culture.

BACKGROUND: Use of nucleic acid amplification tests (NAATs), such as strand displacement assay (SDA), for the detection of gonococcal infection in low prevalence populations is controversial because of the likelihood of false positive results. Use of supplementary NAATs with alternative target sites has been recommended for confirmation of primary NAAT results. AIM: To evaluate if SDA reactive specimens for Neisseria gonorrhoeae, which were either culture positive or negative, can be confirmed by alternative target NAATs such as transcription-mediated assays (TMA). METHODS: SDA reactive specimens were retested by TMA using APTIMA Combo 2 (AC2) and APTIMA GC (AGC) assays. Two different methods of specimen preparation were used to test the specimens. In method A, residual extract after SDA was retested and in method B, the original clinical specimen was re-extracted in TMA medium and then retested. Cervical or urethral swabs were requested to confirm the SDA results by culture. RESULTS: By method A, 26/49 (53.1%) of SDA positive specimens were positive by AC2 and/or AGC; 14/27 (51.8%) culture confirmed SDA positive tests were positive by AC2 and/or AGC. By method B, 38/39 (97.3%) SDA positive results were confirmed by both AC2 and AGC. All the 25 culture confirmed SDA positive tests were confirmed by both AC2 and AGC; 5/6 SDA positive tests that were culture negative were confirmed by both AC2 and/AGC. CONCLUSION: Alternative target site NAATs, such as AC2 and AGC, can be used to confirm SDA positive results using the same clinical specimen. There is high concordance between the three NAATs.