A multi-epitopes tandem antigen for five types of human adenoviruses and its application in development of multivalent IgM immunochromatographic strip test.

In China, human adenovirus serotype 3 (HAdV-3), HAdV-7, HAdV-11, HAdV-14, and HAdV-55 are the main prevalent serotypes causing severe acute respiratory diseases and even deaths. To develop multivalent vaccine and diagnostic reagent, a multi-epitopes tandem antigen (META) was designed. Recombinant META was prepared and its humoral immunogenicity, inducing neutralization antibody ability, antigenicity, and reactogenicity were evaluated. A multivalent immunochromatographic strip constructed using the rMETA was evaluated for its sensitivity and specificity in detecting specific IgM antibodies. As a result, the rMETA induced high titers of specific IgG antibodies, with limited abilities of neutralizing multiple HAdVs. It performed both strong antigenicity and reactogenicity. The multivalent immunochromatographic strip recognized specific IgM antibodies against all the 5 types with sensitivities of 87.5% to 95.3%. It performed high specificity of 97.8%. The present study provides both novel idea for developing multivalent vaccine and reagent for point-of-care detection of multiple types of HAdVs.

Development of Rapid and Visual Nucleic Acid Detection Methods towards Four Serotypes of Human Adenovirus Species B Based on RPA-LF Test.

OBJECTIVES: Human adenoviruses (HAdV) are classified as 7 HAdV species, and some serotypes in species B like HAdV 3, HAdV 7, HAdV 21, and HAdV 55 caused severe symptoms, even fatalities. Patients may be misdiagnosed and inadequately treated without reliable and practical methods for HAdV serotyping. Developing rapid, sensitive, and specific diagnostic methods for HAdV is critical. METHODS: Detection methods were established based on a recombinase polymerase amplification (RPA) assay and lateral flow (LF) test. Specific target sequence was screened, targeting which, primers and probes were designed, synthesized, and screened for establishing assay with high amplification efficiency. Primer or probe concentrations and amplification time were optimized. Detection limit, sensitivity, and specificity were evaluated. Results and Conclusions. Simple, sensitive, and specific RPA-LF methods for detection of four serotypes of HAdV together or separately were established, which had detection limits of 10 to 280 copies/reaction comparable to real-time PCR without recognizing other pathogens. The sensitivity and specificity were >92% and >98%, respectively, evaluated by limited clinical samples. The detection can be completed in 25 min without requirement of any instrument except a constant temperature equipment, showing superior detection performance and promising for a wide use in the field and resource-limited area.

Identification of a Ribosomal Protein RpsB as a Surface-Exposed Protein and Adhesin of Rickettsia heilongjiangensis.

Rickettsia heilongjiangensis is an obligate intracellular bacterium that is responsible for far-eastern spotted fever. Surface-exposed proteins (SEPs) play important roles in its pathogenesis. Previous work identified a ribosomal protein RpsB as an SEP by biotin-avidin affinity, a seroreactive antigen, and a diagnostic candidate protein, indicating that it might play an important role in the pathogenesis of rickettsiae. However, in the absence of other evidence, its subcellular location of being surface-exposed was puzzling because ribosomal proteins are located in the cytoplasm. In the present study, the subcellular location of RpsB was analyzed with bioinformatics tools coupled with immunoelectron microscopy. The adhesion ability of RpsB was evaluated by protein microarray and cellular ELISA. Consequently, different bioinformatics tools gave different location predication results. Thus, RpsB was found in the cytoplasma and inner and outer membranes of R. heilongjiangensis by transmission electron microscopy. Protein microarray and cellular ELISA showed that RpsB binds to the host cell surface and its adhesion ability was even stronger than the known adhesin Adr1. In conclusion, RpsB was visually and directly shown for the time to be an SEP of rickettsiae and might be an important ligand and adhesin of rickettsiae. Its roles in pathogenesis warrant further study.

Development of a rapid and visual detection method for Rickettsia rickettsii combining recombinase polymerase assay with lateral flow test.

OBJECTIVES: Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever, which is the most severe spotted fever group (SFG) rickettsiosis. Developing a simple and reliable detection method is required. METHODS: A detection method for R. rickettsii was established based on a recombinase polymerase amplification (RPA) assay and the lateral flow (LF) test. A specific target sequence was screened, and corresponding primers and probes were designed, synthesized, and screened for establishing an RPA assay with high amplification efficiency. Reagent concentrations, amplification time, and loading volume for strip development were optimized. The detection limit, analytic sensitivity and specificity were evaluated. RESULTS: A rapid, visual, sensitive and specific method for the detection of R. rickettsii based on RPA and the LF test was successfully established. The novel method had a limit of detection of 10 to 50 copies/reaction without recognizing other organisms. Analytical sensitivity and specificity were ≥90% and 100%, respectively, as evaluated by animal and simulative human samples. CONCLUSIONS: Using the established method, detection could be completed in 30 min with visually detectable results by the naked eye, without requirement of any instrument except a constant temperature equipment. The technique shows superior detection performance and is promising for wide use in the field as well as resource-limited areas for R. rickettsii detection.

Rapid and Visual Detection of Coxiella burnetii Using Recombinase Polymerase Amplification Combined with Lateral Flow Strips.

Coxiella burnetii, a global-distributed biological warfare agent, is the causative agent of Q fever. Correct diagnosis of Q fever is challenging and developing a fast, simple, and reliable detection method is necessary. In this study, recombinase polymerase amplification (RPA) assay combined with lateral flow (LF) test was developed targeting 23S rRNA gene of C. burnetii Xinqiao strain. Primers and probe were designed and synthesized, with one set with high amplification efficiency screened for establishment of the method. Reaction conditions were optimized. Sensitivity, specificity, and accuracy were evaluated. The established RPA-LF reaction could be completed in 30 minutes by combining RPA at 37°C with LF at room temperature, with visually judged results. The method showed good specificity without recognizing other bacteria evaluated. It detected positive plasmid and genomic DNA at levels of 10 copies/reaction and 7 copies/reaction, respectively, levels comparable to that of real-time quantitative PCR (RT-qPCR) targeting 23S rRNA gene established previously. Both RPA-LF and RT-qPCR were used to detect C. burnetii-infected mouse samples and the results were fully consistent. The method showed superior detection performance and will provide technical support against C. burnetii in resources-limited areas.

Development of a rapid and visual nucleotide detection method for a Chinese epidemic strain of Orientia tsutsugamushi based on recombinase polymerase amplification assay and lateral flow test.

OBJECTIVES: Orientia tsutsugamushi is an obligate intracellular pathogen that causes scrub typhus. Diagnosing scrub typhus remains a challenge, and a sensitive, specific, simple, and rapid diagnostic test is still needed. METHODS: A recombinase polymerase amplification (RPA) assay combined with a lateral flow (LF) test targeting the 56-kDa gene of a Karp-like strain of O. tsutsugamushi was developed and optimized. The detection limits, sensitivity, specificity, and simulative clinical performance were evaluated. RESULTS: Primers and probe were screened to establish the RPA assay, and the reaction conditions were optimized. The detection limit was 10 copies/reaction in detecting plasmid DNA and 12 copies/reaction in detecting genomic DNA. The RPA-LF method could differentiate O. tsutsugamushi from other phylogenetically related bacteria. The sensitivity was 100% and specificity was over 90%, when evaluated using infected animal samples or simulative clinical samples. Furthermore, the method was completed in 20min at 37°C followed by a 3-5min incubation at room temperature for the development of an immunochromatographic strip, and the results could be determined visually. CONCLUSIONS: This method is promising for wide-ranging use in basic medical units considering that it requires minimal instruments and infrastructure and is highly time-efficient, sensitive, and specific for diagnosing scrub typhus.