Multiplex detection of eight different viral enteropathogens in clinical samples, combining RT-PCR technology with melting curve analysis

Abstract

Background Early and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests. Methods We developed a real time RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. Results Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction among the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The limit detection was ranged from 1 × 102 to 1 × 105 nucleic acid copies/ml for each diarrhea virus. Conclusion The RRCMC method is a suitable rapid clinical test for infectious viruses, with the advantages of high-throughput, low cost, high sensitivity and specificity.

Analysis of the epidemiological characteristics between 2004 and 2017 and prediction of the changing pattern of other infectious diarrhea (OID) under COVID-19 in China

“…[ 10 ] Second, With the continuous improvement of pathogen detection ability, especially the rapid PCR diagnosis technology has been widely used in hospitals at all levels in China, and the diagnostic sensitivity and specificity of OID have been greatly enhanced. [ 11 , 12 ] The improvement in the level of infection diagnosis has led to an increase in the number of reported cases. Third, the network reporting system of infectious diseases (in China) is becoming more and more perfect, and the quality of infectious disease reporting is improving.…”

Molecular Point‐of‐Care Assay Development: Design and Considerations

“…According to Li et al (2022), multiple viruses and bacteria can be detected in one PCR tube through RT-PCR. This is a very important factor because it can prevent the abuse of unnecessary antibiotics and facilitate targeted treatment in early stages of infection (Li et al, 2022). Sensitivity and specificity are high after nucleic acid extraction (Li et al, 2022).…”

“…Analytical sensitivity and specificity are also important measures with distinct differences from clinical sensitivity and specificity, particularly when evaluating infectious disease MDx. Analytical sensitivity, also referred to as the Limit of Detection, defines the lowest (Griffiths et al, 2018;Li et al, 2022;Sun et al, 2022;Torretta et al, 2021;van der Zee et al, 2016) Nasopharyngeal swabs (NPS)/oropharyngeal swabs (OPS), 4°or 2°C to 8°C, 4 to 5 days, in VTM (Yilmaz Gulec et al, 2021); lyophilized pre-mixed reactions, room temperature, 30 days; stool and urine, short-term 2°to 8°C, long-term -20°to -80°C (Sun et al, 2023) concentration of a desired analyte that the assay can detect (MediaLab, n.d.). This value is commonly determined by end-point dilution (MediaLab, n.d.).…”

“…Among the most common symptoms present in these kinds of infections are diarrhea and vomiting, which is very common in children. According to Li et al (2022), multiple viruses and bacteria can be detected in one PCR tube through RT-PCR. This is a very important factor because it can prevent the abuse of unnecessary antibiotics and facilitate targeted treatment in early stages of infection (Li et al, 2022).…”