1 A high-inhibitor tolerance extract-free probe-based qPCR method

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2 based on a novel B-family DNA polymerase for detecting African

3 swine fever virus

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4 Rong Xiang1#, Yanru Wang1#, Guangyi Liu1, 2, Yi Hou3, Song-Qing Hu1*

6 1 School of Food Science and Engineering, South China University of Technology,

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7 Guangzhou, 510640, China

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8 2 Guangzhou Enzyvalley Biotech Co., Ltd, Guangzhou, 510555, China

9 3 State Key Laboratory of Pulp and Paper Engineering, South China University of
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10 Technology, Guangzhou, 510640, China
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11

12 # Rong Xiang and Yanru Wang contributed equally to this work.

13 *Corresponding author: Song-Qing Hu

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14 Address: School of Food Sciences and Engineering, South China University of

Technology, Guangzhou 510641

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15

16 Email: fesqhu@scut.edu.cn
17
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4812529
 18 ABSTRACT

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19 African swine fever, a viral hemorrhagic disease of pigs caused by African swine

20 fever virus (ASFV), is a major danger of food safety. To develop a rapid and sensitive

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21 extract-free probe-based qPCR method for detecting ASFV, a recombinant DNA

22 polymerase (KUpF) was developed by semi-rational engineering based on KOD DNA

23 polymerase, Pfu DNA polymerase, and the 5'-nuclease pFEN1 from Pyrococcus

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24 furiosus. The chimeric KUpF DNA polymerase, especially after chemical modification,

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25 exhibited high DNA amplification performance and 5'-3' exonuclease activity which

26 was necessary for TaqMan qPCR, and showed higher thermal stability and inhibitor
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27 tolerance than Taq DNA polymerase. Meanwhile, an extract-free probe-based qPCR
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28 system was developed for ASFV detection with good performance based on the novel

29 KUpF DNA polymerase, with detection limits as low as 0.25 pg/μL ASFV, and the

30 intra- and inter-assay coefficient of variation were less than 2% in the presence of 0.5%
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31 whole blood. A total of 96 simulated samples were detected, and the sensitivity and

specificity of method were both 100% in the present of 0.5% whole blood, which
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32

33 indicated that KUpF DNA polymerase should be excellent candidate for the application

34 in extract-free probe-based qPCR for ASFV detection.

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35 Keywords: B-family DNA polymerase; inhibitor tolerance; extract-free probe-based

36 qPCR; semi-rational engineering; chemical modification.

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 37 1. Introduction

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38 African swine fever, caused by the African swine fever virus (ASFV), posses

39 highly contagious and 100% fatality rate, is a devastating disease of domestic and wild

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40 pigs (P.-f. Wang, et al., 2022). ASFV has broken out in 45 countries worldwide since

41 2020 and caused more than 2 million death cases (H. Wang, et al., 2023). There is no

42 effective vaccine and antiviral drug, thus a rapid and sensitive detection method is

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43 urgently needed to suppress the spread of the virus. Currently, Real-time PCR is the

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44 standard gold method for ASFV detection, playing a very positive role in the early stage

45 of the ASFV outbreak (X. Wang, et al., 2020). However, as the developing epidemic,
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46 the Real-time PCR method becomes more and more unable to meet the high-frequency
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47 and large-scale requirements of detecting ASFV because it requires additional extract

48 operations (Jiahao Li, et al., 2022). The extraction-free probe-based qPCR simplifies

49 the nucleic acid extraction, and reduces loss of nucleic acid template, all of which are
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50 more in line with the application requirements for pursuing rapid detection in the

context of global infectious disease (Domnich, et al., 2021). The full potential of
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51

52 extract-free probe-based qPCR is limited by the low inhibitor tolerance and low

53 stability of Taq DNA polymerase because of the presence of inhibitors in complex

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54 biological samples, and additional nucleic acid extraction reagents (Wu, et al., 2019).

55 Therefore, a new nucleic acid diagnostic DNA polymerase with excellent specificity,
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56 good stability, and strong inhibitor tolerance is urgently needed.

57 Previous studies have shown that B-family DNA polymerases exhibit better
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58 performance in complex templates amplification and thermostability than A-family

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 59 DNA polymerase (Masashi Miura, Chihiro Tanigawa, Yoshito Fujii, & Satoshi Kaneko,

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60 2013). According to reports, the inhibitor tolerance of DNA polymerase is also related

61 to processivity and elongation rate (Y. Wang, et al., 2004). Among B-family DNA

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62 polymerases, the enzyme from Thermococcus kodakarensis (KOD DNA polymerase)

63 and Pyrococcus furiosus (Pfu DNA polymerase) are more widely used. KOD DNA

64 polymerase has higher processivity and elongation rate, and has superior performance

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65 in PCR and qPCR compared with Pfu DNA polymerase, while Pfu DNA polymerase

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66 shown better thermal stability than KOD DNA polymerase, because of its unique

67 disulfide bond conformation (Elshawadfy, et al., 2014). However, B-family DNA

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68 polymerases are unable to be employed in probe-based qPCR due to a lack of 5'-3'
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69 exonuclease activity.

70 One of the most effective strategies in enzyme engineering is a construction of

71 chimeric proteins derived from natural enzymes and protein domains with desirable
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72 properties. Previous research has found that the fusion with helix–hairpin–helix (HhH)

structural domain could significantly improve the thermal stability of DNA

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73

74 polymerases (Pavlov, Pavlova, Kozyavkin, & Slesarev, 2012). The attachment of

75 DNA-binding proteins (Sto7d) also enhanced the inhibitor tolerance of chimeric

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76 polymerases in loop-mediated isothermal amplification to several of the most common

77 DNA sample contaminants (Oscorbin, et al., 2017). Intriguingly, Bst DNA polymerase
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78 exhibited 5'-3' exonuclease activity and detect nucleic acid after fusing with FEN1 (Ye,

79 Wang, Li, Fang, & Kong, 2021). Therefore, it is feasible to construct a chimeric B-
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80 family DNA polymerase with high amplification performance, thermal stability with

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 81 5'-3' exonuclease activity for extract-free probe-based qPCR assay.

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82 In this study, a chimeric DNA polymerase with DNA amplification and 5'-3'

83 exonuclease activity based on the KOD and Pfu DNA polymerase was constructed. The

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84 recombinant protein (KUpF DNA polymerase) was expressed, purified, and modified

85 by chemical modification. The thermal stability and PCR performance of chimeric

86 DNA polymerase was evaluated. Finally, an extract-free probe-based qPCR assay was

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87 developed for the detection of ASFV based on the KUpF DNA polymerase, and its

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88 specificity, sensitivity, and repeatability were evaluated.

89 2. Materials and methods er

90 2.1. Protein recombination
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91 Firstly, KOFU DNA polymerase was constructed based on KOD DNA

92 polymerase. Compared with wild-type KOD DNA polymerase, the sequence of KOFU

93 DNA polymerase has the following changes: three amino acid residues (Ala, Ser, Ala)
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94 were inserted in sequence after the first methionine residue, and the 355-587 amino acid

sequence were replaced with 332-564 amino acid sequence of Pfu DNA polymerase.
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96 Then, to obtain the 5'-3' exonuclease activity, KUpF DNA polymerase was constructed

97 by fusing with 5'-nuclease named pFEN1, obtained from Pyrococcus furious, with the
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98 linker "SGGGSGGGGSGGGGS" (sequences were shown in the Appendix Table S1)

99 at the C-terminal of KOFU DNA polymerase. Besides, Mut-FP primer and Mut-RP
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100 primer were used to generate D164N, and E166Q mutations (Nishioka, et al., 2001).

101 The target gene were cloned into pET-28a vectors by Hipro DNA Assembly Cloning
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102 kit (EnzyValley, China). Finally, the recombinant plasmids were completely sequenced

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 103 to ensure the fidelity. The schematic representation and amino acid sequence of

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104 recombinant enzyme (KUpF DNA polymerase) were shown in Fig. 1A and Table. S2.

105 2.2. Expression and purification

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106 The BL21 (DE3) strain of E. coli cells harboring the encoding KUpF DNA

107 polymerase plasmid were grown to OD600 reached 0.6-0.8 at 37 °C. Expression was

108 induced by the addition of isopropyl-D-thiogalactopyranoside (IPTG) up to 0.1 mmol/L

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109 concentration. After induction for 10 h at 25 °C, the cells were harvested by

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110 centrifugation at 8,000 × g for 10 min at 4 °C (Avanti JXN-26, USA). The cell pellets

111 were resuspended in buffer A (containing 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 5%

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112 glycerol, pH 8.0) and sonicated for 30 min at 4 °C, followed by centrifugation (20,000
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113 × g, 30 min, 4 °C). The resulting cell supernatant was incubated for 30 min at 75 °C,

114 and centrifugated at 20,000 × g for 30 min at 4 °C to obtain a clarified lysate. Lysate

115 was loaded onto a 1 mL HisTrapTM HP column (GE Healthcare, UK) equilibrated with
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116 buffer A. The recombinant protein was step eluted with buffer B (containing 50 mmol/L

Tris-HCl, 500 mmol/L imidazole, 50 mmol/L NaCl, 5% glycerol, pH 8.0). Fractions

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117

118 containing target protein were collected and dialyzed against buffer C (containing 50

119 mmol/L Tris-HCl, 0.1 mmol/L Ethylenediaminetetraacetic acid disodium salt (EDTA),
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120 50% glycerol, pH 8.0) overnight at 4 °C.

121 Single-stranded E-probe was incubated with chimeric protein to determine

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122 whether the purified polymerase still possessed 3'-5' exonuclease activity (Song, Zhang,

123 & Zhao, 2011). The fluorescence signals were collected every 40 s for 40 cycles to
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124 record the real-time progress. The feasibility of recombinant enzyme was verified by

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 125 detecting plasmids in probe-based qPCR reaction (CFX96 Touch™). The parameter

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126 values of the reaction system are listed in Table S4. The protocols were established: 95

127 °C, 10 min, 40 cycles of amplification (95 °C, 10 s; 60 °C, 30 s).

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128 2.3 Polymerase activity

129 The fluorescence-based assay in conjunction with primer extension procedure was

130 used to evaluate the polymerase activity of DNA polymerase (Driscoll, Rentergent, &

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131 Hay, 2014). The reactions were performed in a 10 μL system comprising 1 × reaction

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132 buffer (120 mmol/L Tris-HCl, 2.5 mmol/L MgCl2, 20 mmol/L KCl, 10 mmol/L

133 (NH4)2SO4, 0.025% TritonX-100, pH 7.8). 60 nmol/L Test-1 primer and Test-2 primer
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134 (Table S1), 20 μmol/L dNTPs, and 0.8 × SYBR Green I. By incubating Test-1 primer
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135 with Test-2 primer at 65-50 °C (-5 °C/30 s), a 5'-overhang flap were created, which was

136 filled with a saturating quantity of dNTPs. 0.4 μL of recombinant enzyme or KOD FX

137 Neo (TOYOBO, Japan) were added to start the reaction. The fluorescence signals were
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138 collected every 10 s for 30 cycles. Fluorescence intensity increased linearly of the

reaction was selected to calculate the initial slope to calculate polymerase activity
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139

140 (Nasiri & Nasiri, 2018). While the pH optimum of the enzyme was investigated by

141 measuring the polymerase activity at a range of 7.2-10.0.

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142 2.4 PCR efficiency

143 The PCR efficiency was determined according to the method of previous research
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144 with slight modifications (Cho et al. 2012). The extension efficiency was calculated

145 from the 1 kb λ DNA synthesized in different extension times (1, 10, 30, and 60 s,
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146 respectively). Each optimal amount of purified KUpF, KOD, Pfu, and KOFU DNA

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 147 polymerase were added to a 20 μL PCR system (1 ng of λ DNA, 200 nmol/L each of

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148 the λ-FP primer, and λ-RP primer, 200 μmol/L of dNTPs). PCR procedures were set as

149 follows: 94 °C, 3 min; 35 cycles of amplification (98 °C, 10 s; 55 °C, 30 s; 72 °C for

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150 different extension times). The effect of ion concentration (0-125 mmol/L NaCl) on

151 DNA polymerase was carried out in a 20 μL reaction system comprising 10 ng of

152 pUC19, 200 nmol/L each of the 19S-FP primer and 19S-RP primer, 200 μmol/L of

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153 dNTPs. PCR procedures were set as follows: 94 °C for 3 min, 35 cycles of amplification

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154 (98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s, respectively), and a final extension

155 for 5 min. The PCR products were subjected to electrophoresis on 1.5% (v/v) agarose
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156 gel for analysis.
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157 2.5 Steady-state kinetic analyses

158 The steady-state kinetic analyses of KUpF, KOD, Pfu, and KOFU DNA

159 polymerase were assayed with 0 - 1 μmol/L pre-annealed primer extension substrates
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160 by using the polymerase activity assay. Each reaction system contained 2.5 nmol/L

polymerases, and 200 mmol/L dNTPs. The initial rate of each reaction was plotted
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161

162 against substrate concentration and fitting to the equation according to Y. Wang, et al.

163 (2004).
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164 2.6 Chemical modification of KUpF DNA polymerase

165 Chemical modification of KUpF DNA polymerase was performed as the

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166 procedure described by Dixon and Perham (1968). KUpF DNA polymerase was diluted

167 to 1 μmol/L using 40 mmol/L Tris-HCl, pH 9.0. Citraconic anhydride was dissolved
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168 with N, N-dimethylformamide (Sigma, USA), and added dropwise to KUpF DNA

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 169 polymerase at a molar ratio of 2,000:1. The mixture was ultrafiltered into dialysis buffer

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170 (50 mmol/L Tris-HCl, 0.1 mmol/L EDTA, and 50% glycerol, pH 9.0) after being

171 incubated at 25 °C for 3 h.

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172 2.7 Stability assay

173 Thermostability of DNA polymerase was assessed using the previously described

174 methodology with minor modifications (Cho, Kim, Lee, Youn, and Kwon (2012). 50

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175 U/mL of DNA polymerase was initially incubated at 95 °C and 98 °C for 0 - 6 h, and

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176 incubated at 30-90 °C for 2 h, respectively. The data for thermal stability were fitted to

177 the equation according to Y. Wang, et al. (2004). For pH stability, the DNA polymerase
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178 was incubated at 100 mmol/L PBS (pH 5.0-7.5) and 100 mmol/L Tris-HCl (pH 7.5-
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179 10.0) buffers for 12 h. The residual polymerase activity was measured, and the relative

180 activity was calculated and plotted versus time, with the polymerase activity without

181 treatment set to 100%.

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182 2.8 Fluorescence spectroscopy

Fluorescence spectroscopy was measured with an RF-6000 fluorescence

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184 spectrophotometer (Tokyo, Japan) equipped with a xenon lamp source and a 1.0 cm

185 quartz cell (Xia et al., 2021). The sample was diluted to 0.1 mg/mL with 10 mmol/L
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186 PBS (pH 7.4). The excitation wavelength (slit width, 3 nm) was set to 295 nm, and the

187 emission spectra was scanned over the spectra range of 300 to 450 nm. Protein surface
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188 hydrophobicity was assessed using the previously described methodology with a little

189 change (Xia et al., 2021). 20 μL of 4 mmol/L 1-Anilinonaphthalene-8-sulfonic acid

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190 (ANS) and 2 mL DNA polymerase with a concentration range of 0.1 to 0.5 mg/mL

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 191 were mixed, then stand for 15 min in the dark. The fluorescence intensity was detected

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192 at an excitation wavelength of 370 nm and an emission wavelength of 470 nm. The

193 widths of both excitation slit and emission slit were set at 3 nm. The spectrum of the

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194 blank reference of the corresponding buffer should be subtracted from each sample.

195 Three parallel measurements were made on each sample.

196 2.9 Inhibitor tolerance

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197 To evaluate the inhibitor tolerance of DNA polymerase, the probe-based qPCR

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198 was performed with 1 U/μL of hot-start KUpF DNA polymerase and Taq DNA

199 polymerase. The reaction system (10 μL) was composed of 1 ng of pMD-18T-nCoV-N
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200 plasmid, 200 nmol/L each of the primers and probes, and 200 μmol/L of dNTPs. The
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201 effect on amplification efficiency of KUpF DNA polymerase with different inhibitors

202 including NaCl, ethanol, Sodium dodecyl sulfate (SDS), EDTA, urea, bile salt, heparin

203 sodium, tannic acid, and whole blood in the reaction system at various concentrations
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204 were examined.

2.10 Applications of KUpF DNA polymerase for extract-free probe-based qPCR

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205

206 method

207 To demonstrate the applicability of KUpF DNA polymerase, ASFV genomic

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208 DNA was detected in the present or absence of blood samples. The lower limits of

209 detection and reproducibility were determined using serial dilutions of ASFV genomic
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210 DNA (25, 5, 2.5, 0.5, 0.25, and 0.05 pg/μL) as templates, and nuclease-free water was

211 used as the negative control. In parallel, the intra- and inter-assay reproducibility were
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212 measured using the above template in triplicate, and the coefficients were calculated

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 213 using the formula of the geometric mean Ct values. The sensitivity and specificity were

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214 evaluated by testing 48 simulated negative samples and 48 simulated positive samples.

215 The simulated sample was prepared as follows: whole blood collected from healthy

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216 pigs was diluted to 2.5% (v/v) by PBS as the negative sample, and extracts of infected

217 blood samples were diluted 32-256-fold using 2.5% (v/v) healthy whole blood as

218 positive sample. The reaction was performed in a 20 μL volume containing 1× reaction

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219 buffer (120 mmol/L Tris-HCl, pH 7.8, 2.5 mmol/L MgCl2, 20 mmol/L KCl, 10 mmol/L

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220 (NH4)2SO4), The final reaction contained 200 nmol/L each of primers and probes, 200

221 μmol/L dNTPs, and 2 μL of template DNA. The reaction condition was as follows:
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222 95 °C for 10 min; 45 cycles at 95 °C for 10 s, 60 °C for 30 s.
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223 3. Results and discussion

224 3.1 Construction of KUpF DNA polymerase

225 According to previous research, B-family DNA polymerases have superior

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226 thermostability, inhibitor tolerance, and proofreading properties (M. Miura, C.

Tanigawa, Y. Fujii, & S. Kaneko, 2013). In exception to the lack of 5'-3' exonuclease
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228 domain, the 3'-5' exonuclease activity of B-family DNA polymerase may lead to the

229 degradation of primers and probes, which might play a negative role in qPCR detection
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230 (del Prado, et al., 2018; Dodd, et al., 2020). To allow the B-family DNA polymerase to

231 be used in probe-based qPCR, the mutation and pFEN1 protein were introduced into
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232 KOFU DNA polymerase (Hosfield, Mol, Shen, & Tainer, 1998; Nishioka, et al., 2001).

233 The chimeric KUpF DNA polymerase was designed by semi-rational engineering as
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234 Fig. 1A, and was successfully constructed, expressed, and purified (Fig. 1B). To obtain

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 235 high thermal stability and high amplification, the amino acid sequence of the

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236 recombinant proteins (KOFU DNA polymerase and KUpF DNA polymerase) consists

237 of the the 3'-5' nucleic acid exonuclease and thumb domain of KOD, with the palm and

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238 fingers domain of Pfu DNA polymerase. Additionally, the site mutation was performed

239 (D164N, E166Q, E170H, F175S, M182T, A240V, E608K, V660A, V670D, G788A)

240 to eliminate 3'-5' exonuclease activity of DNA polymerase, which prevented the free

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241 probe from being hydrolyzed by the recombinant enzyme (Fig. 1C). The pFEN1 protein

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242 was fused and allowed the polymerase to obtain 5'-3' exonuclease activity, and a probe-

243 based qPCR reaction was successfully triggered by the recombinant enzyme alone (Fig.
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244 1D). By constructing the chimeric enzyme with high stability, amplification
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245 performance, and 5'-3' exonuclease activity, it is possible to introduce B-family DNA

246 polymerases into a highly specific mode of hydrolytic probe detection.

247 3.2 Stability of KUpF DNA polymerase

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248 For nucleic acid need to denature at high temperature, a DNA polymerase with

high thermal stability in PCR/qPCR is need (Pavlov, Pavlova, Kozyavkin, & Slesarev,
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250 2004). After preincubation at 95 °C or 98 °C, the relative polymerase activity of KUpF

251 DNA polymerase was measured. The calculated half-life at 95 °C and 98 °C for KUpF
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252 DNA polymerase are 250 and 70 min, respectively (Fig. 2A, Table S4). It’s important

253 to note, the half-life at 95 °C of Pfu DNA polymerase and KOD DNA polymerase were
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254 360 min and 720 min, respectively (Takagi, et al., 1997). The palm-fingers domain of

255 Pfu DNA polymerase and KOD DNA polymerase contains two disulfide links that keep
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256 the protein structurally stable (Killelea & Connolly, 2011). The alignment results of

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 257 predicted 3D structure of KUpF DNA polymerase (generated using RoseTTAFold) and

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258 Pfu DNA polymerase showed that the disulfide bond located on the α helix in Pfu may

259 not have formed in KUpF DNA polymerase, which may result in decreased

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260 thermostability (Fig. S1A). The hypothesis had been further confirmed by measuring

261 the free sulfhydryl content of different enzymes under the same molar concentration. It

262 was found that the KUpF DNA polymerase has the highest concentration (4.6 mmol/L)

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263 of free sulfhydryl than KOD, Pfu DNA, and KOFU DNA polymerases (Fig. S1B). The

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264 relative polymerase activity of KUpF DNA polymerase was gradually decreased when

265 the temperature was higher than 50 °C (Fig. 2B). Although the thermostability of KUpF
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266 DNA polymerase was unexpectedly weaker than the parent polymerase, it still showed
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267 superior thermal stability than Taq DNA polymerase, which half-life at 95 °C was only

268 40-50 min (Y. Wang, et al., 2004).

269 The effect of pH condition on polymerase activity of DNA polymerase was

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270 evaluated by preincubating DNA polymerase with different buffers. The optimal pH of

KUpF DNA polymerase was 8.0 (25 °C) in Tris-HCl buffer (Fig. 2C). KUpF DNA
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272 polymerase exhibited more than 60% polymerase activity after incubated for 12 h at

273 different pH conditions (Fig. 2D).

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274 3.3 Effect of chemical modification on KUpF DNA polymerase

275 Chemical modification of enzyme is considered as a distinct way to improve

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276 biocatalytic properties, which can also be used to block polymerase activity (Giri, Pagar,

277 Patil, & Yun, 2021). The fundamental mechanism is amino acid residues bind to acid
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278 anhydrides, resulting in inhibiting polymerase activity (Hassani, 2012). To evaluate the

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 279 effect of chemical modification on DNA polymerase, the enzymatic properties and

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280 surface hydrophobicity of polymerase were investigated. Chemical modification led to

281 a remarkable increase in the half-life of KUpF DNA polymerase (Fig. 2A, Table S4),

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282 and the relative polymerase activity of KUpF DNA polymerase still retained above 80%

283 of after incubation at 90 °C for 2 h (Fig. 2B). These results suggested that citraconic

284 anhydride successfully improved the thermal stability of KUpF DNA polymerase. At

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285 the same time, KUpF DNA polymerase exhibited the higher pH stability after modified

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286 by chemical modification, it was found that hot-started KUpF DNA polymerase could

287 maintain better stability in a wider range of pH (pH 5.0-7.0) (Fig. 2C, 2D).
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288 Meanwhile, the endogenous fluorescence and surface hydrophobicity of KUpF
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289 DNA polymerase were measured to determine the effect of chemical modification on

290 the structure of KUpF DNA polymerase. Endogenous fluorescence measurements

291 revealed that the fluorescence intensity of recombinant enzyme decreased after
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292 chemical modification, and the maximum emission wavelength moved from 330 to 333

nm (Fig. 2E). The phenomenon indicated that the tryptophan residues of KUpF DNA
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294 polymerase were more easily exposed to the solvent after chemical modification. At the

295 same time, the surface hydrophobicity of the chemically modified polymerase was
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296 reduced (Fig. 2F). It might because the modification of ε-NH2 on lysine residues

297 changed the positive charge of the lysine residues to a negative charge, and resulted in
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298 a net anionic charge, which reduced isoelectric point of KUpF DNA polymerase.

299 Previous research have found the surface charges of proteins is key parameters that can
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300 dramatically alter their biophysical and kinetic properties (Paik, Bhadra, & Ellington,

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 301 2022). These results suggested that citraconic anhydride successfully improved the

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302 stability of KUpF DNA polymerase by changing the surface charge of KUpF DNA

303 polymerase.

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304 The specificity is impressionable in extraction-free TaqMan qPCR because of the

305 large number of exogenous components. Hot-start DNA polymerase may be carried out

306 to avoid this issue by employing antibody or chemical modifications. However, the cost

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307 of antibody modification is quite significant, and the polymerase activity is difficult to

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308 completely block at times (Stevens, Appleby, & Kennedy, 2016). Because of the

309 negative effect on the 3'-5' exonuclease activity of B-family DNA polymerases,
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310 chemical modification is mainly applied in the modification of A-family DNA
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311 polymerases, although it has the advantage of being cheap and can completely block

312 polymerase activity (Dronina, Bubniene, & Ramanavicius, 2021). In this study, the

313 successful chemical modification of KUpF DNA polymerase provides a paradigm for
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314 the immobilization of B-family DNA polymerases by amino or carboxyl groups.

3.4 PCR efficiency of KUpF DNA polymerase

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315

316 To evaluate the effect of additional domain fusion on the efficiency of B-family

317 DNA polymerase in PCR reaction, the PCR efficiencies of KOD, Pfu, KOFU, and
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318 KUpF DNA polymerase were determined. High salt concentration influences the

319 binding interactions between polymerase and template, which will impact PCR
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320 efficiency (processivity and elongation) of polymerase (Gao, et al., 2021). The salt

321 tolerance of DNA polymerase was determined with a 0.4-kb plasmid in multiple
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322 concentration NaCl in PCR. As expected, Pfu DNA polymerase preferred the lowest

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 323 NaCl concentrations (< 50 mmol/L), but is also higher than that of Taq DNA

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324 polymerase (40 mmol/L) (Y. Wang, et al., 2004). At the same time, the efficient

325 amplifications were observed over a broad range of NaCl concentrations with KOD,

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326 KOFU, and KUpF DNA polymerase, and a significant amount of product was observed

327 over 100 mmol/L NaCl (Fig. 3A). The results indicated that KOD DNA polymerase

328 and its derivatives can bind nucleic acids with high-concentration salt solution.

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329 Meanwhile, a steady-state kinetic analysis was performed to investigate the DNA

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330 binding affinity between DNA polymerase and nucleic acid. KOFU DNA polymerase

331 and KUpF DNA polymerase had significantly lower Km (DNA) compared to Pfu or
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332 KOD DNA polymerase, which meant chimeric protein had a stabilizing effect on the
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333 interactions between polymerase and template. But the fusion of pFEN1 may reduce

334 the synthesis rate of KUpF DNA polymerase since a decrease of kcat within the fusion

335 protein (Table S5). This phenomenon might be explained that the increase of binding
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336 sites between the template and polymerase for the fusion of pFEN1, which reduced the

rate of the dissociation of KUpF DNA polymerase from the template, affecting the
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337

338 synthesis efficiency and leading to the decrease of its amplification performance

339 (Pavlov, Belova, Kozyavkin, & Slesarev, 2002).

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340 DNA polymerases with higher extension efficiency, which may lead to shorter

341 extension times to amplify the same target (Wang et al. 2004). With purified KUpF,
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342 KOD, and KOFU DNA polymerase, a 1 s/cycle extension time was sufficient to amplify

343 a 1 kb target. However, the 60 s/cycle extension time was a prerequisite for Pfu DNA
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344 polymerase (Fig. 3B), suggesting that the PCR efficiency of the KUpF DNA

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 345 polymerase was much higher than the Pfu DNA polymerase. These results confirmed

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346 that domain swapping can retain the desired functional features of chimeric enzyme,

347 and enhance the DNA binding affinity, which could lead to increased tolerance and

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348 processivity.

349 3.5 The inhibitors tolerance of different DNA polymerase

350 The inhibitor tolerance of DNA polymerase determines whether it can be used in

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351 extraction-free probe qPCR system, because many inhibitors have adverse effects on

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352 qPCR reactions, which may impact the sensitivity and accurate of pathogen detection

353 (Jiaxuan. Li, et al., 2023). In here, the inhibitor tolerance of KUpF DNA polymerase
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354 and Taq DNA polymerase in real-time qPCR reaction was compared to evaluate the
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355 feasibility of KUpF DNA polymerase applied in extraction-free probe qPCR system. It

356 was shown that KUpF DNA polymerase was significantly more resistant to seven

357 inhibits than that of the Taq DNA polymerase. It's worth noting that KUpF DNA
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358 polymerase exhibited excellent detection performance in qPCR reaction, which still

successfully detected template under 1% whole blood, while Taq DNA polymerase
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359

360 completely lost its activity exceeds 0.1%. The reason may be that the mutation and

361 attachment of pFEN1 enhanced the affinity between polymerase and template, which
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362 improved the tolerance of KUpF DNA polymerase to the inhibitor (Hosfield, et al.,

363 1998). At the same time, the use of B-family DNA polymerase retained higher inhibitor
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364 tolerance. Taken together, it is feasible to construct an extraction-free probe qPCR

365 system with KUpF DNA polymerase.

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 366 3.6 Detection performance of KUpF DNA polymerase for extract-free probe-based

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367 qPCR method

368 Direct amplification with whole blood would avoid several problems, such as long

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369 time, high cost, and loss of nucleic acids. However, heme, IgG and anticoagulants in

370 whole blood can impact the detect efficiency of qPCR reaction. Improving the inhibitor

371 tolerance of DNA polymerase can reduce the effect of PCR inhibitors on the

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372 amplification reaction. In this paper, the whole blood tolerance of hot-start KUpF was

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373 analyzed, and the results are shown in Fig 5. The maximum tolerance of KUpF DNA

374 polymerase to whole blood can reach 9% (v/v) (not detected at 10% (v/v)), which is
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375 much higher than that of wild-type Taq DNA polymerase (Fig. S2). However, due to
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376 the fluorescence signal was affected by the hemoglobin and Fe3+ in whole blood, which

377 is more suitable for detection in the PCR system rather than qPCR reaction (Lee, et al.,

378 2023). Therefore, the 0.5% (v/v) whole blood addition were selected to evaluate the
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379 efficiency of the subsequent qPCR assay by the extraction-free probe method.

The limits of detection of serially diluted extracted ASFV genome DNA detection
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380

381 in presence of 0.5% whole blood was analyzed and provided in Table 1. As expected,

382 KUpF DNA polymerase successfully amplified the ASFV genomic DNA as low as 0.25
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383 pg/μL, while Taq DNA polymerase completely lost polymerase activity, indicating that

384 KUpF DNA polymerase has the potential for DNA detection in whole blood samples.
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385 The coefficient of variation (CV) of experiments was 2%, indicating the good

386 reproducibility of the method. Meanwhile, Trombley Hall, McKay Zovanyi,

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387 Christensen, Koehler, and Devins Minogue (2013) evaluated the ability of nine

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 388 commercially qPCR reagents for direct TaqMan qPCR detection, and qPCR reagents

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389 could only amplify target gene in 0.5% whole blood. But the fluorescence signal

390 intensity was very low, and unreproducible. The excellent performance of KUpF DNA

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391 polymerase at low starting templates may be due to its higher affinity for template, and

392 that this is more suitable for the amplification of low starting copy number nucleic acids.

393 The specificity and sensitivity results shown that KUpF can perform accurate

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394 differential diagnoses, which sensitivity and specificity were both 100% for target

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395 pathogens (Table 2, Table S6). These results show that this method, without false

396 positives, which is a basic requirement in infectious disease diagnosis and control, and
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397 provide the support that recombinant enzyme could be used in clinical. Taken the
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398 sensitivity, specificity, and reproducibility together, KUpF DNA polymerase should be

399 the most promising enzyme in extraction-free probe qPCR system.

400 4. Conclusion
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401 In this study, a novel chimeric protein (KUpF DNA polymerase) was constructed

based on pFEN1 protein, KOD, and Pfu DNA polymerase with site mutation (D164N,
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402

403 E166Q, E170H, F175S, M182T, A240V, E608K, V660A, V670D, G788A). After

404 chemical modified by citraconic anhydride, KUpF DNA polymerase exhibited

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405 excellent thermal, pH stability, and inhibitor tolerance, while it showed good extension

406 efficiency in PCR reaction and detection performance in the extraction-free probe
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407 qPCR assay. These proved that it is possible to expand the application of B-family DNA

408 polymerase by adding 5'-3' exonuclease domains. Therefore, KUpF DNA polymerase
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409 provided the possibility to develop simple and robust probe-based qPCR detection,

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 410 which can eliminate the cumbersome steps in extracting DNA and have good detection

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411 prospects for detecting nucleic acid from blood samples. However, the research only

412 stays on the detection of simulated samples, large-scale clinical trials are needed to

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413 further evaluate the performance of the method.

414 Declaration of competing interest

415 The authors declare that they have no known competing financial interests or

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416 personal relationships that could have appeared to influence the work reported in this

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417 paper.

418 Acknowledgements er
419 This research was supported by the Guangdong Basic and Applied Basic Research
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420 Foundation, China (grant number 2021A1515220141).

421

422 Supplementary materials

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423 Supplementary material associated with this article can be found in the online version.
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 424 References

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434 Dixon, H. B., & Perham, R. N. (1968). Reversible blocking of amino groups with

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474 thermostability of chimeric Bst DNA polymerase for isothermal amplification

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477 commercially-available DNA polymerases for direct PCR. Revista Do
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 490 Paik, I., Bhadra, S., & Ellington, A. D. (2022). Charge Engineering Improves the

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496 13510-13515.

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497 Pavlov, A. R., Pavlova, N. V., Kozyavkin, S. A., & Slesarev, A. I. (2004). Recent

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499 efficient applications. Trends in Biotechnology, 22(5), 253-260.
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507 Stevens, A. J., Appleby, S., & Kennedy, M. A. (2016). Many commercial hot-start

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511 Oka, M., & Imanaka, T. (1997). Characterization of DNA polymerase from

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513 Environmental Microbiology, 63(11), 4504-4510.

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518 Wang, H., Sun, Y., Zhou, Y., Liu, Y., Chen, S., Sun, W., Zhang, Z., Guo, J., Yang,

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519 C., Li, Z., & Chen, L. (2023). Unamplified system for sensitive and typing

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 534 1197-1207.

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535 Wu, Y. H., Wei, T., Zhang, X. T., Zhao, Y. Q., Wang, J. K., Cong, L., Xu, B. Z., &

536 Shao, X. Q. (2019). Development and evaluation of a direct TaqMan qPCR

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537 assay for the rapid detection of diverse carnivore amdoparvoviruses. Mol Cell

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539 Ye, X., Wang, N., Li, Y., Fang, X., & Kong, J. (2021). A high-specificity flap probe-

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540 based isothermal nucleic acid amplification method based on recombinant

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541 FEN1-Bst DNA polymerase. Biosensors & Bioelectronics, 192.

542 er
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 543 Figure captions

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544 Fig. 1 Analysis of KUpF DNA polymerase. (A) Schematic representation of KUpF

545 DNA polymerase. (B) SDS-PAGE analysis of KUpF DNA polymerase. (M: Marker;

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546 Lane 1: Total bacterial proteins; Lane 2: Supernatant after cell disruption; Lane 3:

547 Supernatant after heat treatment; Lane 4: Flowthrough fraction of chromatography;

548 Lane 5: Elution fraction). (C) 3'-5' exonuclease activity of the recombinant enzyme. (D)

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549 Probe-based qPCR reaction using the recombinant enzyme.

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550 Fig. 2 Thermostability (A, B), optimal pH (C), pH stability (D), endogenous

551 fluorescence (E) and surface hydrophobicity (F) of KUpF DNA polymerase before and
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552 after chemical modification.
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553 Fig. 3 Comparison of the salt tolerance (A) and extension efficiency (B) of KOD, Pfu，

554 KOFU, and KUpF DNA polymerase. Lanes 1-6: 0, 25, 50, 75, 100, and 125 mmol/L

555 NaCl.
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556 Fig. 4 Inhibitor tolerance of KUpF DNA polymerase and Taq DNA polymerase. (A)

NaCl; (B) Ethanol; (C) SDS; (D) EDTA; (E) Urea; (F) Bile salt; (G) Whole blood; (H)
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557

558 Heparin sodium; (I) Tannic acid

559
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560

561
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 562 Fig. 1

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563
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564
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 565 Fig. 2

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A 5 B 120
KUpF
HS-KUpF
4

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100

Relative activity（% ）
ln(relative activity)

80

KUpF(95℃)
60
KUpF (98℃)
1
HS-KUpF (95℃)
HS-KUpF(98℃)

v
0 40
0 1 2 3 4 5 6 -20 0 30 40 50 60 70 80 90
Time (h) Temperature (℃)

C 120
D 120

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KUpF
HS-KUpF
100 100
Relative activity (%)

Relative activity (%)

80 80

60 60

40

20
er 40

20
KUpF(PBS)
KUpF(Tris-HCl)
HS-KUpF(PBS)
HS-KUpF(Tris-HCl)
0 0
7.0 7.5 8.0 8.5 9.0 9.5 10.0 5 6 7 8 9 10
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pH （25 ℃） pH (25℃)

E 2500
F 400 a
KUpF
Surface hydrophobicity Intensity(H0)

HS-KUpF
2000
300
Fluorescence Intensity

1500

200
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1000
b
100
500
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0 0
350 400 450 HS-KUpF KUpF
Wavelength (nm)

566 A

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 568 Fig. 3

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 570 Fig. 4

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A 35
B 26
C 30
HS-KUpF HS-KUpF HS-KUpF
HS-Taq HS-Taq HS-Taq
a a 28 a
a abc
ab
30 24 abc abc
a ab ab c
bc c 26
c a
Ct

Ct

Ct
b b bc
24 b b c d
25 22 e

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bb cc c
d e 22

ND ND ND ND ND ND ND ND ND ND ND ND ND
20 20 20
0 25 50 75 100 125 0 1 2 3 4 6 8 10 0 0.01 0.02 0.03 0.05 0.075 0.1 0.15

NaCl (mmol/L) Ethanol（%） SDS（‰）

D 35
E 40 a F 30
HS-KUpF a HS-KUpF HS-KUpF
HS-Taq HS-Taq HS-Taq
b 28
35 a
30
26
Ct

Ct

Ct
30 a
a
bc bc b bc
24
bc b
25 bc a c b c d
b d c cd bc a

v
bc bc b 25 b d d
b d
c d d 22
c
NDND ND ND ND ND ND ND ND ND ND
20 20 20
0 0.5 1 1.5 2 2.5 3 4 5 0 100 200 400 450 475 500 600 0 0.5 1 1.5 2 3 4

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EDTA (mmol/L) Urea (mmol/L) Bile salt (μg/μL)
G H 45 I 30
45
HS-KUpF HS-KUpF a HS-KUpF
a HS-Taq
HS-Taq a HS-Taq
40 40 28

a
35 35 26 a
b

Ct
Ct

Ct

b b a
b 24 c cd cd
30 b c 30 d
e
d c c b
25 f e 25 d d d d 22
g c e

20
0 0.05 0.1

Whole blood （%）

0.25
ND
0.5
ND
0.75
ND
1
ND ND ND
1.5
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20
0 0.005 0.01

Heparin sodium （U/μL）

0.015 0.02 0.03
ND ND ND
0.04
20
0 25 50
ND
100

Tannic acid (ng/μL)

ND
200
ND ND
400 600
ND NDND
750

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 573 Table 1 Repeatability test of qPCR

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Intra-assay variability Inter-assay variability
Template concentration (pg/μL)
Ct±SD CV (%) Ct±SD CV (%)
25 31.1±0.14 0.44 31.33±0.29 0.92

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5 33.55±0.17 0.52 33.42±0.13 0.40
2.5 35.33±0.15 0.43 35.08±0.2 0.57
0.5 37.26±0.26 0.70 37.13±0.13 0.35
0.25 39.29±0.29 0.75 38.46±0.63 1.64

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No template control ND ND ND ND

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574 ND: No amplification was detected
575
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 576 Table 2 Sensitivity and specificity of simulated samples

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True-Positive False-Negative Sensitivity specificity

Positive 48 0
100% 100%

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Negative 0 48

577

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