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Molecular detection of HPV DNA is considered as the gold standard for the diagnosis of cervical disease. Although the molecular assays for the identification of HPV16 and HPV18 have helped identify cervical cancer incidents, they are restricted to specialized laboratories. Thus, we developed a novel 2-stage, nested-like nucleic acid amplification method, named IsoPCR, to amplify the E6 gene of HPV16 and HPV18 with high analytical sensitivity and specificity. The performance of IsoPCR was compared to that of conventional PCR and LAMP. The analytical sensitivity of IsoPCR (1 copy/test) was 10-fold higher than conventional PCR and 25-fold higher than conventional LAMP. IsoPCR displayed significant amplification specificity (100%) and efficiency, as well. In conclusion, IsoPCR is a highly sensitive and specific diagnostic tool and it is suitable for the detection of low copy number of viral DNA in clinical specimens, providing critical information to healthcare providers., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RT-PCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 5'NCR (subgrouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Modern molecular approaches in Human Enterovirus detection rely on the designing of generic and often degenerate primers in order to amplify specific sequences within the enterovirus genome. In the present study a comparative application of primer sets targeting 5'UTR, the VP1 region, the 3D region as well as a long genomic fragment including the 3'end of VP1, the full length of 2A and 2B, and the 5' moiety of the 2C-coding region was attempted, in order to evaluate their specificity and suitability. The best amplification results from the investigation of 21 CAV reference strains, all six CBV reference strains and 44 clinical strains varying in origin and time of isolation, arose using primer sets 292-222 and UC53-UG52. Based on the above results we conclude that some of the published protocols need to be improved so as to fulfill the demands of an accurate detection and typing of Coxsackie A and B viruses. Contrarily, two of the protocols applied were proved to be more accurate in terms of specificity and general applicability, suggesting that RT-PCR followed by a simple RFLP assay in the case of primer pair UC53-UG52 or by sequencing and sequence analysis in the case of primer set 292-222 should constitute alternative means of modern typing and diagnostics against conventional immunological classification methods.

In the present report, the results of a comparative study in the detection of all Echoviruses reference strains as well as of 38 clinical isolates are presented. Using RT-PCR with already published primer pairs (UG(52)-UC(53), 292-222, 012-011 and EUG2a, 2b, 2c-EUC2) from the 5'UTR, the VP1 region as well as a long genomic fragment including the VP1 3' end, the entire coding sequence of 2A, 2B, and the 5' moiety of the 2C-coding region amplification was effective with all reference and clinical Echovirus isolates with primer pair UG(52)-UC(53) while with 292-222 and 012-011 were amplified 27/28 reference Echovirus strains and all clinical isolates. As far as EUG2a,2b,2c-EUC2 is concerned, the RT-PCR gave a positive result for 26/28 reference Echovirus strains and 34/38 clinical isolates. The sequence analysis of a large part of the 5'UTR has revealed that there is no correlation between 5'UTR identity and the currently recognized human enterovirus species. It has been suggested that part of VP1 coding sequence would correlate well with serotype since a number of important neutralization epitopes, as well as receptor recognition sequences, lie within the VP1 coding sequence. Therefore, UG(52)-UC(53) and 292-222 primer pairs seem to be the most appropriate for Echovirus detection and, moreover, UG(52)-UC(53) is useful for the classification of enteroviruses into genetic clusters (sub-groups) while 292-222 for the identification of enteroviruses by amplicon sequencing.