Without adaptive immunity, invertebrates have evolved innate immune systems that react to antigens on the surfaces of pathogens. These defense mechanisms are included in horseshoe crab hemocytes’ cellular responses to pathogens. Secretory granules, large (L) and small (S), are found on hemocytes. Once the invasion of pathogens is present, these granules release their contents through exocytosis. Recent data in biochemistry and immunology on the granular constituents of granule-specific proteins are stored in large and small granules which are involved in the cell-mediated immune response. L-granules contain most clotting proteins, which are necessary for hemolymph coagulation. They also include tachylectins; protease inhibitors, such as cystatin and serpins; and anti-lipopolysaccharide (LPS) factors, which bind to LPS and agglutinate bacteria. Big defensin, tachycitin, tachystatin, and tachyplesins are some of the essential cysteine-rich proteins in S-granules. These granules also contain tachycitin and tachystatins, which can agglutinate bacteria. These proteins in granules and hemolymph act synergistically to fight infections. These biomolecules are antimicrobial and antibacterial, enabling them to be drug resistant. This review is aimed at explaining the biomolecules identified in the horseshoe crab’s hemolymph and their application scopes in the pharmaceutical and biotechnology sectors.
A viral transport medium (VTM) was developed following the Centers for Disease Control and Prevention, USA (US-CDC) standard operating procedure (SOP) DSR-052-05 with necessary improvisation and was used for storing coronavirus disease 2019 (COVID-19) swab specimens. Considering Bangladesh’s supply chain and storage conditions, improvisation was essential for extending sample storage time while retaining efficiency. In-house VTM was produced using Hank’s balanced salt solution (HBSS) supplemented with 1% bovine serum albumin V (BSA), 0.5 µg /mL of gentamicin sulfate, and 100 µg/mL of fluconazole. The produced VTM composition, quality, sterility, specificity, and efficiency were verified in-house and through an independent contract research organization (CRO). An accelerated stability study projected that under the recommended temperature (4 °C), it would remain stable for four months and preserve samples for over a month. The real-time reverse transcriptase–polymerase chain reaction (rRT-PCR) test detected the targeted N gene and ORF1ab gene from the VTM stored samples. Our VTM is equally as effective as the Sansure Biotech VTM in keeping SARS-CoV-2 RNA specimens detectable in rRT-PCR (100% sensitivity and specificity in random and blinded samples). In conclusion, the BRiCM VTM will make the battle against pandemics easier by effectively collecting and storing nasopharyngeal and oropharyngeal swabs for COVID-19 detection.
In this study, we evaluated the performance of the in-house developed rRT-PCR assay for SARS-CoV-2 RNA targeting the envelope (E) and nucleocapsid (N) genes with internal control as human RNase P. A total of 50 positive samples and 50 negative samples of SARS-CoV-2 were tested by a reference kit at site 1 and a subset (30 positives and 16 negatives) of these samples are tested blindly at site 2. The limit of detection (LoD) was calculated by using a replication-deficient complete SARS-CoV-2 genome and known copy numbers, where Pseudo-virus samples were used to evaluate accuracy. On site 1, among the 50 SARS-CoV-2 positive samples 24, 18, and eight samples showed high (Ct < 26), moderate (26 < Ct ≤ 32), and low (32 < Ct ≤ 38) viral load, respectively, whereas in site 2, out of 30 SARS-CoV-2 positive samples, high, moderate, and low viral loads were found in each of the 10 samples. However, SARS-CoV-2 was not detected in the negative sample. So, in-house assays at both sites showed 100% sensitivity and specificity with no difference observed between RT PCR machines. The Ct values of the in-house kit had a very good correlation with the reference kits. LoD was determined as 100 copies/mL. It also displayed 100% accuracy in mutant and wild-type SARS-CoV-2 virus. This Bangasure™ RT-PCR kit shows excellent performance in detecting SARS-CoV-2 viral RNA compared to commercially imported CE-IVD marked FDA authorized kits.
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