Identification And Typing Of Human Coronaviruses And Botulinum Neurotoxin Based On Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF MS)is wildly used in the detection and identification of bacteria,viruses and biotoxins in the field of biosecurity due to its high sensitivity,high resolution and high throughput.In this study,we established a highly sensitive method for detecting human coronavirus(HCoV)and botulinum neurotoxin(BoNT)based on MALDI-TOF MS,which integrated the application of mass spectrometry technology in the field of biosafety.At present,there are seven CoVs that infect humans,among which HCoV-229E,HCoV-OC43,HCoV-NL63 and HCoV-HKU1 can only cause common respiratory diseases with mild symptoms and self-healing,and will not pose a huge threat to the public.However,severe acute respiratory syndrome virus(SARS-CoV),Middle East respiratory syndrome virus(MERS-CoV),and severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)cause human-to-human acute respiratory infections.These three diseases are highly contagious,lethal and fatal.Initial symptoms of HCoV infection are similar,but treatments vary widely.Rapid and accurate detection of HCoVs is not only conducive to timely diagnosis and prescribing the right medicine in the early stage of infection,but also is of great significance for the prevention and control of large-scale epidemics of acute respiratory infectious diseases.In this study,we combined multiplex polymerase chain reaction(m PCR)with MALDI-TOF MS,and designed primers and probes based on single nucleotide polymorphism(SNP)sites to construct a method that can simultaneously detect seven HCoVs,named HCoV-MS.We selected N and Rd Rp as the detection targets of HCoV-229E,HCoV-NL63,HCoV-OC43 and HCoV-HKU1,N,E and ORF1b as the detection targets of SARS-CoV,and N,Rd Rp,E and ORF1b as the detection targets of MERS-CoV as the detection target,N1,N2,ORF1b,S and S(D614G)were used as the detection target of SARS-CoV-2 to design primers and probes to complete the construction of the system.The specificity of HCoV-MS was verified by plasmids and m PCR cycles with other common respiratory viruses,and the sensitivity of HCoV-MS was verified by using gradient concentrations of plasmids.Ultimately,the HCoV-MS limit of detection was 1?5copies/reaction.HCoV-MS was used in the United Nations Secretary-General's Investigative Mechanism(UNSGM)Laboratory External Quality Assessment Activity(EQAE-CoV-2020)organized by the Robert?Koch Institute in Germany in 2020.We used this method to accurately identify all HCoVs in the assessment samples(including difficult samples).In addition,151 clinical samples were tested,and the test results were all consistent with real-time quantitative PCR(q PCR),and a total of 42 D614G mutants were detected.The sensitivity and specificity of HCoV-MS and q PCR were compared by the detection of 26serially diluted positive samples,and it was found that the specificity and sensitivity of the HCoV-MS method were basically the same as those of q PCR.The HCoV-MS detection system is small and requires a small amount of sample,only 1?L of which is needed for the reaction.It also has high throughput,and 384 samples can be completed within 30 minutes.The HCoV-MS has advantages in micro-sample and large-scale detection,which is expected to be a supplement to q PCR technology,and may also provide ideas for the identification of other viruses and further typing of mutant strains.BoNT is the most toxic biological toxin known so far,mainly produced by Clostridium botulinum.There are currently 9 serotypes,A?H and X.The serotypes associated with human poisoning are A,B,E and F,of which A and B are the main ones.Common types of poisoning are foodborne,iatrogenic,traumatic and infantile botulism.In recent years,various types of processed food have been developed one after another,the quality of food varies,and the population of eating out has increased sharply,resulting in frequent occurrence of C.botulinum food poisoning.In addition,because the lethal dose of BoNT is extremely low and relatively easy to prepare,it is used by some international terrorist forces and extremist organizations to create panic,posing a huge threat to human peace and security.Therefore,establishing a rapid and accurate identification and typing method for BoNT is a necessary condition for optimizing treatment and reducing mortality,and is also of great significance for maintaining national biosecurity.The light chain of BoNT is a Zn²⁺dependent protease that specifically cleaves specific proteins required for neural signaling.Using the enzymatic activity of BoNT,we constructed a method for the identification and typing of BoNT/A and BoNT/B by MALDI-TOF MS,named BoNT-MS.The identification and typing are realized by enrichment of toxin enzyme cleavage substrate peptides by magnetic bead-coupled antibodies and isotope-labeled internal standard method.The optimum reaction system of BoNT-MS was determined to be p H7.2 Hepes buffer containing 50?M Zn Cl₂,10m M DTT,1mg/m L HSA,and 100?M substrate peptide.In addition,we also explored the detection of different complex matrix samples,and finally found that the lowest detection amounts of BoNT/A-MS and BoNT/B-MS in PBS matrix samples were 2×LD₅₀ and 40×LD₅₀,respectively.The limit of detection of BoNT/B-MS and BoNT/B-MS in plasma matrix samples were 2×LD₅₀ and 4×LD₅₀,respectively.The limit of detection of BoNT/A-MS and BoNT/B-MS in milk matrix samples were 2×LD₅₀ and 4×LD₅₀,respectively,and BoNT/A-MS and BoNT/B-MS in sandy soil matrix samples have the limit of detection of2×LD₅₀ and 80×LD₅₀,respectively.We used BoNT-MS to participate in the UNSGM laboratory external quality assessment activity(EQAE-Biotoxins-2021)organized by the Robert?Koch Institute in Germany in 2021,and realized the evaluation of multiple complex matrix samples(serum,milk,sand and soil)in the identification of BoNT.In conclusion,the BoNT-MS method established in this study can rapidly identify and type BoNT/A and BoNT/B,which is expected to be a supplement to mouse bioassay.In conclusion,MALDI-TOF MS has increasingly become an indispensable detection and identification method,which can play an important role in the detection and identification of HCoVs and BoNT/A and BoNT/B,and is suitable for large-scale screening of samples.It is of great significance to the national and military biosafety construction.