Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients

The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (−2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70–80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.

Identification of MFLP fingerprint for higher seed zinc accumulation in barley DH population

Selection through molecular markers for seed Zn accumulation might be an efficient complementary breeding tool in barley breeding. To develop a specific molecular markers, 150 DH lines derived from a cross between Clipper (low-Zn-accumulator) and Sahara-3771 (high-Zn-accumulator) were screened under field and glasshouse conditions. Microsatellite-anchored fragment length polymorphism (MFLP) fingerprint generated by SSR-anchor primer MF128 in combination with AFLP primer MseI-AGA (5?-GATGAGTCCTGAGTAAAGA-3?) was identified as a candidate marker for tagging seed Zn accumulation gene. The sequencing of the band showed a marker of 369 bp with the sequence of SSR anchor primer MF128 and MseI-AGA at the two ends as expected. The MFLP marker associated with higher seed Zn accumulation has potential to be converted to a simple, sequence-specific, PCR-based, low-cost marker amenable to large populations, making it potentially viable for marker-assisted selection in barley breeding. This marker might be useful in the improvement of barley productivity and nutritional quality in Zn-deficient environments.

Macroarray Detection of Plant RNA Viruses Using Randomly Primed and Amplified Complementary DNAs from Infected Plants

Membrane-based macroarrays provide a relatively inexpensive technology with the potential to detect hundreds of pathogens in a single assay. For the simultaneous detection of a large number of pathogens, it is necessary to obtain sufficient nucleic acids for labeling, and any amplification reactions need to be performed using unbiased, pathogen-non-specific primers. A nonradioactive macroarray system is described to test for plant RNA viruses using 70-mer oligonucleotide probes immobilized on nylon membranes. Starting with a total plant RNA extract, complementary DNA (cDNA) and second-strand syntheses were carried out using an anchor primer sequence with random pentamers coupled at the 3′ end. Subsequent synthesis by polymerase chain reaction using the anchor primer alone resulted in a relatively unbiased amplification of plant and viral RNAs. These cDNAs were chemically labeled and the product used as a target in hybridization analyses. The system was validated using RNA extracts from plants infected with Cucumber mosaic virus, Potato virus Y, and Potato leaf roll virus (PLRV). Despite the relative excess of host-derived nonviral sequences, viral RNAs were amplified between 100- and 1,000-fold and were detected in single and mixed infections. The macroarray sensitivity was comparable to that of double-antibody sandwich enzyme-linked immunosorbent assay, with PLRV being detected in sap dilutions of 1:100. The potential for the development of a relatively inexpensive multipathogen detection system is discussed.

Flanking-Sequence Exponential Anchored (FLEA) PCR - a Sensitive and Highly Specific Method for Detecting Retroviral Integrant-Host-Junction Sequences.

Retroviral vectors integrate in the host cell genome and are widely used for gene therapy. Since integration is largely random, each site represents a unique integration event and can be used to study clonal evolution of engrafted cells. Integration can however be deleterious, and has been associated with oncogenesis. To date, PCR methods for studying the integrant-host junction (IHJ) have been laborious, unspecific and difficult to perform on complex samples. We have developed a method that is simpler and highly specific. We generate libraries of integrant-host junctions which can then be resolved by sequencing. The approach works even for analyses of highly complex samples. 1) Linear PCR is performed on sample DNA with biotinylated primer annealing to the 5′ end of the LTR. The subsequent longer biotinylated linear PCR fragments which contain IHJ or internal junctions are separated from genomic DNA with streptavidin paramagnetic beads. 2) We add an anchor primer with a known 5′ sequence and several degenerate 3′ bases. 3) A complimentary strand from this primer to the LTR is generated with T7 DNA polymerase. 4) A blocking oligo is added to the polymerase mixture, which anneals just before the 3′LTR of the internal junction and prevents the complementary strand from the internal sequence being generated, so that >90% of the anchored DNA contains external flanking genomic sequence. 5) We PCR amplify the material from a nested primer in the LTR to a primer, which anneals to the known sequence of the anchor primer. 6) Finally, a smear is generated which contains randomly anchored IHJs. FLEA-PCR was designed so that the most 5′ 33bp of the LTR (which contain an NheI site) are unprimed allowing easy validation of PCR product. Sequencing of these smears reveals that >99% of these DNA fragments contain this 33bp of 5′ extremity and are preceded by genomic sequences. With blocking, 90% of sequences are external IHJs. To validate the technique, 6 HeLa cell clones containing a single integrant were studied. The 5′ IHJ was characterized by FLEA-PCR amplification. The 3′ IHJ was successfully amplified by PCR with a primer designed on prediction from the human genome sequence and the 4bp repeat of junctional sequences was evident in all HeLa clones. Clonable smears can be generated with samples containing <0.1% transduced cells. The mean length of flanking genomic sequence is 97bp (ranging from 0 to 674bp). Digestion of smears amplified with a 6FAM-labelled LTR primer with CviJI and capillary electropheresis analysis allows precise counting of sample complexity. More accurately, random sequencing of smear can be assembled by custom written software. It is possible to study samples with over 80 different integrants in a single experiment. There are several advantages of using FLEA-PCR over restriction digest based methods. There is less PCR artifact. Since integrants yield varying fragments of random length, there is no PCR bias caused by differing amplicon lengths. This is of particular benefit when a short internal band can "swamp" the reaction. Secondly, no bias is accrued due to the frequency of occurrence of a particular restriction site. Finally, the technique is highly compatible with automation and high-throughput sequencing. This approach should facilitate IHJ analysis for assessing both retroviral safety and the biologic fate of transduced cells.

Dexamethasone enhances ras-recision gene expression in cultured murine fetal lungs: role in development

We have shown that dexamethasone (Dex) accelerates maturation and differentiation of cultured fetal murine lungs (Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Surg Forum 47: 692–695, 1996). We now demonstrate that although Dex inhibits thinning of acinar walls and secondary septa formation, it does, however, promote lung growth. CD-1 murine fetal lungs were cultured for 7 days in the presence and absence of 10 nM Dex. Dex-modulated genes were investigated and identified by differential display of mRNAs performed with specific anchor primer H-T11G and 24 arbitrary primers. Thirty-five differentially expressed cDNAs were isolated, subcloned, sequenced, and identified through BLAST searches. One of these cDNAs, termed Dex2, with enhanced expression in Dex-treated lungs, had 100% similarity with ras-recision gene ( rrg), also known as the lysyl oxidase ( LOX) gene that encodes lysyl oxidase. LOX gene is very highly conserved, with significant sequence similarity among mouse, rat, and human. Two other cDNAs, termed Dex1 and Dex4, were also identified as rrg, with 92 and 97% sequence similarity with the existing data bank sequence of rrg. LOX enzyme is known to downregulate p21 ras protein and play a central role in the maturation of collagen and elastin in the extracellular matrix as well as modulate the cytoskeletal elements. Thus LOX may be important in lung developmental processes involving epithelial-mesenchymal interactions.