{'fraction_non_alphanumeric': 0.029425999419223695, 'fraction_numerical': 0.01664892072403446, 'mean_word_length': 4.873791927231381, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Simultaneous detection of Marburg virus and Ebola virus with TaqMan‐based multiplex real‐time PCR method

Abstract Background Marburg virus (MARV) and Ebola virus (EBOV) are acute infections with high case fatality rates. It is of great significance for epidemic monitoring and prevention and control of infectious diseases by the development of a rapid, specific, and sensitive quantitative PCR method to detect two pathogens simultaneously. Methods Primers and TaqMan probes were designed according to highly conserved sequences of these viruses. Sensitivity, specificity, linear range, limit of detection, and the effects of hemolysis and lipid on real‐time qPCR were evaluated. Results The linearity of the curve allowed quantification of nucleic acid concentrations in range from 103 to 109 copies/ml per reaction (MARV and EBOV). The limit of detection of EBOV was 40 copies/ml, and MARV was 100 copies/ml. It has no cross‐reaction with other pathogens such as hepatitis b virus (HBV), hepatitis c virus (HCV), human papillomavirus (HPV), Epstein‐Barr virus (EBV), herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Repeatability analysis of the two viruses showed that their coefficient of variation (CV) was less than 5.0%. The above results indicated that fluorescence quantitative PCR could detect EBOV and MARV sensitively and specifically. Conclusions The TaqMan probe‐based multiplex fluorescence quantitative PCR assays could detect EBOV and MARV sensitively specifically and simultaneously.


Filoviruses such as Marburg virus (MARV) and Ebola virus (EBOV)
can cause severe hemorrhagic fevers in humans and primates, and as one of the most dangerous pathogens for humans listed by the World Health Organization (WHO). 1 MARV and EBOV are mainly transmitted by direct contact and aerosol, 2,3 the incubation period is commonly 3-9 days, the elderly can be more than 2 weeks.
A variety of body fluids, such as secretions, excretions, contaminants, and environments from patients with hemorrhagic fever and animals infected with MARV or EBOV can infect a variety of immune cells through damaged skin, eyeballs, nasal passages, and oral mucosa. 4 Patients can present with serious bleedings and hemorrhagic shock syndrome. 5 It has high pathogenicity and fatality rate.
MARV was first discovered in Marburg, Germany in 1967 and was named Marburg virus according to the location of this disease.
It was the first filovirus found in human. 6 MARV is a member of the filoviruses family, has caused outbreaks in sub-Saharan Africa, and can cause severe disease with a high case fatality rate. Humanto-human transmission may occur in a home or hospital setting. 7 Although MARV was discovered more than 50 years ago, there is no effective treatment has yet been developed, 8 except the vaccine has a preventive effect. 9 The first outbreak of the EBOV occurred in the Ebola River region in southern Sudan and Zaire (is now the democratic republic of Congo region) in 1976, patient mortality rates as high as 90%. 10 In the outbreak in West Africa between 2013 and 2016, there were about 28,000 cases were confirmed and 11,000 deaths were reported, indicating the high mortality rate of this disease. 11 Although the viruses have only been endemic in some African countries, 12 and no large-scale virus-infected patients have been reported in other regions, there is a potential risk of virus transmission owing to countries around the world interact more and more with each other and the movement of people and goods increases.
Moreover, MARV and EBOV can be used as a potential biological terrorist weapons or biological agents to use. 13 Therefore, it is necessary to establish a rapid and specific laboratory test method for the early diagnosis and prevention of MARV and EBOV infection.
TaqMan-based real-time fluorescence quantitative polymerase chain reaction (PCR) has the characteristics of rapid, sensitive, specific, and high throughput, and has been used in the detection of a variety of viruses. [14][15][16] MARV and EBOV has a negative-strand RNA of approximately 19 kb, respectively. This RNA genome contains seven genes, include nucleoprotein (NP), virion protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, and the RNA-dependent RNA polymerase L. 2,17 Each one of these genes encodes a single protein product. 18,19 We aimed to establish a rapid, sensitive TaqMan-based real-time fluorescence quantitative PCR detection assays according to the genetic sequence of the viruses, to provide technical support for laboratory testing of prevention and control of these severe infectious diseases.

| Identification of sequences and design of primers and probes
According to the whole genome sequence of 42 strains of MARV published by GenBank, DNASTAR software was used for multiple sequence alignment (MSA) to screen the highly conserved NP gene of MARV nuclear protein as the target gene. According to the whole genome sequence of EBOV published by GenBank, DNASTAR software was used for multiple sequence alignment to screen out the highly conserved NP gene of EBOV as the target gene.
Primer Premier 6.0 software was used to design specific primers and probes that met multiple response conditions. Multiple primers were designed with the following default settings: primer melting temperature (T m ) set at 60°C approximately. TaqMan probes for EBOV and MARV were labeled at the 5′-end with the reporter molecule: hexachloro-6-carboxy-fluorescein (HEX), pentamethine cyanine (CY5), respectively. And the TaqMan probes were labeled at the corresponding 3′-end with the quenchers: 6-carboxy-tret ramethyl-rhodamine (TAMRA), 8-Bromo-7-hydroxyquinoline 2 (BHQ2) (Sangon Biotech Co., Ltd.), respectively. And the size of PCR product of EBOV and MARV were 163 and 180 bp, respec-

| Gene synthesis and preparation of standard substance positive template
The highly conserved sequences of MARV NP gene and EBOV NP gene were synthesized artificially as the target genes and cloned into pUC57 vector through gene synthesis. The gene synthesis was completed by Sangon Co., Ltd.
The cloned strain of pUC57 was cultured in E. coli and plasmid pUC57 was extracted. The absorbance (A) of plasmid at wavelengths of 260 and 280 nm was measured by spectrophotometer, and purity was determined according to the ratio of A260/A280. The plasmid concentration was determined and converted to copy number of plasmids according to the following formula. Dissolve a certain amount of plasmid in the blood and calculate the template concentration and copy number in the blood according to the following formula.
Finally, the template was stored at −20°C for reserve.

| Optimization of fluorescence quantitative PCR reaction system and reaction conditions
TaqMan-based real-time PCR was performed on the Lightcycler 480 fluorescence quantitative PCR system (Roche Diagnostics). First, a single system qualitative reaction was performed for each pathogen. On this basis, the primer concentration, probe concentration, and T m of the multiple fluorescence quantitative PCR reaction were optimized to establish the optimal reaction system for simultaneous detection of two viruses.

| Sensitivity test
The plasmid of EBOV and MARV nucleic acid concentrations 1.0 × 10 1 , and 1.0 × 10 0 copies/ml were prepared in batches and each template was tested three times. Real-time fluorescence quantitative PCR was used for detection and statistical analysis to determine its sensitivity.

| Specificity test
The specificity of the assays was verified by the existence or absence of amplification using MARV template nucleic acid corresponding to EBOV primers and probe or vice versa. In addition, potential cross-

| Preparation of standard curve
A 10-fold series of diluted plasmids were used as templates for amplification, and the corresponding standard curve was established.

| The results of only MARV detection, simultaneous detection, and only EBOV virus detection of two viruses with various mixtures of synthesized two plasmids
The showed that the set copy number had a good correlation with the corresponding Ct value, and the correlation coefficient R 2 > 0.99 was less than 5.0%. These indicating that this method has good accuracy and repeatability.

| Determination of specificity
The specificity of the assays was verified by the existence or absence of amplification using MARV template nucleic acid

| The effect of lipid and hemolysis on the performance of the assay
There were significant differences of final measured values in the Ⅰ level of EBOV (p = 0.002) and MARV (p < 0.001) affected by different degrees of hemolysis (Table 2). There were significant differences of final measured values in the Ⅰ level of EBOV (p = 0.001) and MARV (p < 0.001) affected by different lipid turbidity (Table 3). There were no significant differences of final measured values in the level Ⅱ, Ⅲ of  The single-tube multiplex PCR method adopted in this study can simultaneously detect EBOV and MARV in one reaction tube. The linearity of the curve allowed quantification of nucleic acid molecules in a range from 10 3 to 10 9 copies/ml per reaction in the assay.

| CON CLUS ION
The TaqMan probe-based multiplex fluorescence quantitative PCR assays could detect EBOV and MARV sensitively, specifically, and simultaneously. This study might provide technical support for laboratory testing of prevention and control of these severe infectious diseases. The further validation of this detection assay and developing multiplex real-time PCR assayable a greater number of dangerous viruses will be the focus of our next work.

ACK N OWLED G EM ENTS
The author would like to thank other colleagues whom were not listed

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
Heming Wu and Zhixiong Zhong designed the study. Heming Wu and Zhikang Yu performed the experiments. Qingyan Huang helped to analyze the data. Heming Wu prepared the manuscript. All authors were responsible for critical revisions, and all authors read and approved the final version of this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.